From sudhee26 from gmail.com Fri Feb 1 00:13:02 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Fri Feb 1 15:48:18 2008 Subject: Increment of mRNA level after siRNA In-Reply-To: <215786.90734.qm@web90404.mail.mud.yahoo.com> References: <215786.90734.qm@web90404.mail.mud.yahoo.com> Message-ID: http://precedings.nature.com/documents/1206/version/1/files/npre20071206-1.doc Sudheendra. On Feb 1, 2008 3:22 AM, Ozan Aygun wrote: > Hi, > > Please check out the following references: > > 1. Long-Cheng Li, Steven T. Okino, Hong Zhao, Deepa > Pookot, Robert F. Place, Shinji Urakami, Hideki > Enokida, and Rajvir Dahiya. Small dsRNAs induce > transcriptional activation in human cells.(2006) PNAS: > 3 October 2006. (doi: 10.1073/pnas.0607015103) > > 2. Garber Ken.Small RNAs Reveal an Activating Side. > (2006)Science:314,741-2. > > This phenomenon is worth to consider and examples are > increasing... > > Perhaps RNA world is much much more complicated than > we think... > > Best, > > Ozan > > > > ____________________________________________________________________________________ > Never miss a thing. Make Yahoo your home page. > http://www.yahoo.com/r/hs > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From Antonio.Sarikas from gmail.com Fri Feb 1 15:27:45 2008 From: Antonio.Sarikas from gmail.com (AS) Date: Fri Feb 1 15:49:54 2008 Subject: CHX chase: what is a good control? Message-ID: I am looking for a cytoplasmatic protein with a halflife of approx 2 hrs to use as a control in a CHX chase. (not as a loading control). My concern is that CHX can kill the cells thereby releasing cytoplasmic proteins while the "cell skeleton" with actin remains, resulting in a decay of your protein of interest, that is actually not real. Thank you for your opinion! From ucgatan from ucl.ac.uk Fri Feb 1 16:09:00 2008 From: ucgatan from ucl.ac.uk (Tom Anderson) Date: Fri Feb 1 21:42:14 2008 Subject: MTT In-Reply-To: <031501c8642a$b35b7aa0$2e7a01a3@dpag.ox.ac.uk> References: <031501c8642a$b35b7aa0$2e7a01a3@dpag.ox.ac.uk> Message-ID: On Thu, 31 Jan 2008, Yvonne Couch wrote: > I have another (relatively stupid) question. This is definitely not my > education but my own brain not being able to understand the problem. > I've just started using the MTT assay to look at the toxicity of two > different compounds. I get how the assay works, mitochondrial reduction > of the salt to formazan which forms purple crystals, more crystals = > more mitochondrial activity. But I apparently need to run a standard > curve with increasing cell numbers in each well. I'm not really sure > why. I've run it and I get more purple as my cell number increases but > surely this is just because there are MORE cells and therefore the very > nice line graph I have doesn't really tell me anything except that more > cells make more crystals? Exactly. I've never done an MTT assay, but i think it's just a really roundabout way of counting cells: if you know how much purple colour you get from ten thousand cells, then when you measure the amount of purple you get from your experimented-on cells, you can work out how many there are. It's a bit like counting a crowd of people by taking off their socks and weighing them. Except you can't actually come up with a real number, because you don't actually know how many cells you have in the control, because you seed them one day, and measure two days later. All you can say is that there are X% as many cells as in the control. Or rather, X% as much mitochondrial reductase activity, which could correspond to a change proliferation rate (affecting cell number), growth rate (affecting cell, and so mitochondrial, mass), regulation of mitochondrial replication or growth (affecting the mass of mitochondria per unit of cell mass), or the level of expression or activity of the reductases. I think if i had access to a Coulter counter, i'd be using that instead, and getting direct measurements of cell number and volume. Or is the point to make a measurement which involves mitochondrial parameters, and so reflects cell health? Or is this just for people who have plate readers and are determined to use them? > The proliferation rate of the cells, no matter what their number, should > essentially be the same? Yes, until they get confluent, or use up their nutrients, or soil their medium or whatever and start growing more slowly. > My next query is that after running my standard curve I'm supposed to > pick a cell number and use that in all the assays. I have picked 10k > cells per well since it was nicely in the middle of my graph but on > different days it has not given the same reading when untreated (0.3 > absorbance one day 0.6 the next) Yep, that sounds like cells to me. > so if I treat the cells with a compound how am I supposed to know if > it's doing anything to their rate of proliferation? You need a standard every time you do the experiment. I'm not sure if you need a whole standard curve, or just a single control well, where you haven't added a test compound. The latter should be fine if the assay's linear. Maybe three control wells, to be sure. tom -- Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT (t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson@ucl.ac.uk From josenet from tiscali.co.uk Fri Feb 1 16:33:56 2008 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Fri Feb 1 21:42:21 2008 Subject: MTT References: Message-ID: <60hhqiF1qkfq4U1@mid.individual.net> "Yvonne Couch" wrote in message news:mailman.441.1201825059.2451.methods@net.bio.net... >I have another (relatively stupid) question. This is definitely not my > education but my own brain not being able to understand the problem. I've > just started using the MTT assay to look at the toxicity of two different > compounds. I get how the assay works, mitochondrial reduction of the salt > to formazan which forms purple crystals, more crystals = more > mitochondrial > activity. But I apparently need to run a standard curve with increasing > cell numbers in each well. I'm not really sure why. I've run it and I > get > more purple as my cell number increases but surely this is just because > there are MORE cells and therefore the very nice line graph I have doesn't > really tell me anything except that more cells make more crystals? The > proliferation rate of the cells, no matter what their number, should > essentially be the same? > > > > My next query is that after running my standard curve I'm supposed to pick > a > cell number and use that in all the assays. I have picked 10k cells per > well since it was nicely in the middle of my graph but on different days > it > has not given the same reading when untreated (0.3 absorbance one day 0.6 > the next) so if I treat the cells with a compound how am I supposed to > know > if it's doing anything to their rate of proliferation? > > > > These seem like fairly obvious questions but I am genuinely confused! > > Cheers > > Y. Well, I've never done one of these tests, but I have reasonable experience quantitating signals (southern, RT, microarrays, etc). The reason to try various amounts of cells is to find a range in which you are confident you can detect differences in... in this case mitochondrial activity. Any system such as this has a useful range. Below a particular threshold, the signal is too low. Above a certain threshold the system is saturated and you don't get a stronger signal when the activity is higher. If you plate different amounts of cells (known) and plot the signal you measure vs. the number of cells, the useful range is found where the relationship is linear. After a certain point, the graph will level... if you use a number of cells in the linear part of teh graph, you can then quantify the difference in mitochondrial activity. Does this make sense to you? (by the way, not a stupid question at all...) This is related to a petty hate of mine... when you see on a paper a Western blot, for instance, and the show you the pattern corrresponfing to whatever antibody, and below some other antibody (such as tubulin, or whatever) as a "loading control" to show that all lanes contain similar amounts of protein... and many are saturated! Well, if you saturate, sure, they will all look the same! Watch out for it, it's *very* common. Usually it doesn't destroy the point the paper makes as they have other evidence, but still... Jose From ucgatan from ucl.ac.uk Sat Feb 2 08:43:41 2008 From: ucgatan from ucl.ac.uk (Tom Anderson) Date: Sat Feb 2 10:49:21 2008 Subject: GFP, PBS and warmth Message-ID: Hi all, Can anyone tell me what my GFP-expressing cells will look like having been fixed in formaldehyde and then incubated in PBS at 37 degrees for twelve hours? I'm guessing they'll be okay, provided they haven't started growing bacteria; i can't see how the incubation in PBS would destroy the GFP or reverse the fixation. I thought i should ask anyway! Note to self: in future, when doing a timecourse experiment, put the coverslips for each timepoint in separate four-well dishes. tom -- Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT (t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson@ucl.ac.uk From z_hattab from yahoo.com Sat Feb 2 04:50:53 2008 From: z_hattab from yahoo.com (dana hattab) Date: Sat Feb 2 15:03:45 2008 Subject: ABI 3130 genetic analyzer Message-ID: <10225.20398.qm@web58215.mail.re3.yahoo.com> Hi everyone: i want to perform a gene scan useing the device ABI 3130 genetic analyzer, i will use the dyes FAM and HEX and ROX 400 as a size standard. my problem is i want to know which filter set is found on the machine (i used to use the ABI 3100, is it the same) i asked the person who work with ABI, but since he is knew he dose not know. so how can i know the virtual filter sets fitted in our device, and if they are compatible with the above dyes? thanx ____________________________________________________________________________________ Looking for last minute shopping deals? Find them fast with Yahoo! Search. http://tools.search.yahoo.com/newsearch/category.php?category=shopping From aawara from pontiff-playground.org Sat Feb 2 21:44:46 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Sun Feb 3 14:41:55 2008 Subject: CHX chase: what is a good control? References: Message-ID: In , AS wrote: > > I am looking for a cytoplasmatic protein with a halflife of approx 2 > hrs to use as a control in a CHX chase. (not as a loading control). My > concern is that CHX can kill the cells thereby releasing cytoplasmic > proteins while the "cell skeleton" with actin remains, resulting in a > decay of your protein of interest, that is actually not real. > How about d2EGFP? It is a derivative of EGFP with a PEST sequence that reduces its half-life to about 2 hours. Clontech used to sell it. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From wxfhome from gmail.com Sun Feb 3 22:29:15 2008 From: wxfhome from gmail.com (WANG XF) Date: Sun Feb 3 23:18:44 2008 Subject: How to prepare 1M H2O2 using 30% hydrogen peroxide? Message-ID: <404daa2f0802031929ne677896q566a0ea84761e93c@mail.gmail.com> Hello! I want to use H2O2 as an inducer of apoptosis. How to prepare 1M H2O2 using 30% hydrogen peroxide? Thank you all! Lao WANG From ben.long from yourfinger.anu.edu.au Sun Feb 3 23:49:22 2008 From: ben.long from yourfinger.anu.edu.au (Bean Long) Date: Mon Feb 4 11:21:41 2008 Subject: How to prepare 1M H2O2 using 30% hydrogen peroxide? In-Reply-To: References: Message-ID: <47a69952$1@clarion.carno.net.au> WANG XF wrote: > Hello! > > I want to use H2O2 as an inducer of apoptosis. How to prepare 1M H2O2 using > 30% hydrogen peroxide? > > Thank you all! > > Lao WANG Here's a handy little link: http://www.trimen.pl/witek/calculators/stezenia.html From aawara from pontiff-playground.org Mon Feb 4 07:38:46 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Mon Feb 4 11:21:53 2008 Subject: How to prepare 1M H2O2 using 30% hydrogen peroxide? References: Message-ID: In , WANG XF wrote: > I want to use H2O2 as an inducer of apoptosis. How to prepare 1M H2O2 using > 30% hydrogen peroxide? The molecular weight of H2O2 is 34 daltons (or close enough to 34). 1M H2O2 = 34 g/L 30% H2O2 = 300 g/L Do the math, and dilute it in ddH2O. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From hroychow from nmsu.edu Mon Feb 4 16:02:07 2008 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Mon Feb 4 16:11:48 2008 Subject: How to prepare 1M H2O2 using 30% hydrogen peroxide? In-Reply-To: <404daa2f0802031929ne677896q566a0ea84761e93c@mail.gmail.com> References: <404daa2f0802031929ne677896q566a0ea84761e93c@mail.gmail.com> Message-ID: <2988.128.123.174.0.1202158927.squirrel@webmail.nmsu.edu> 30% H2O2 is 300g/Liter 1M H2O2 is 34g/Liter (approx) Will that suffice? (This is what most of us are amazed about! Apoptosis is a good thing --- thank God!) > Hello! > > I want to use H2O2 as an inducer of apoptosis. How to prepare 1M H2O2 > using > 30% hydrogen peroxide? > > Thank you all! > > Lao WANG > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Hiranya S. Roychowdhury, Ph.D. Asst. Professor, Health & Public Services Dona Ana Community College New Mexico State University Las Cruces, NM 88003 From sudhee26 from gmail.com Mon Feb 4 14:54:58 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Mon Feb 4 16:11:58 2008 Subject: Post Translational Modification of Proteins Message-ID: Hi, can somebody tell me what changes would occur to the molecular weight of a protein being run in a denaturing PAGE after it undergoes Post translational modification? like sumoylation can increase mol weight by 11kD. Sudheendra. -- Think before agree Think before you nod but STOP thinking and You Are God From pow.joshi from gmail.com Mon Feb 4 17:43:49 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Mon Feb 4 20:36:43 2008 Subject: How to prepare 1M H2O2 using 30% hydrogen peroxide? In-Reply-To: <2988.128.123.174.0.1202158927.squirrel@webmail.nmsu.edu> References: <404daa2f0802031929ne677896q566a0ea84761e93c@mail.gmail.com> <2988.128.123.174.0.1202158927.squirrel@webmail.nmsu.edu> Message-ID: <710764ea0802041443u7f602799h43d435adc31facaf@mail.gmail.com> On 04/02/2008, Dr. Hiranya S. Roychowdhury wrote: > > > > 30% H2O2 is 300g/Liter > > 1M H2O2 is 34g/Liter (approx) > > Will that suffice? > > (This is what most of us are amazed about! Apoptosis is a good thing --- > thank God!) LOL! ... sorry I could'nt help but notice your last remark about apoptosis.... Pow > Hello! > > > > I want to use H2O2 as an inducer of apoptosis. How to prepare 1M H2O2 > > using > > 30% hydrogen peroxide? > > > > Thank you all! > > > > Lao WANG > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Hiranya S. Roychowdhury, Ph.D. > Asst. Professor, > Health & Public Services > Dona Ana Community College > New Mexico State University > Las Cruces, NM 88003 > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From pow.joshi from gmail.com Mon Feb 4 18:00:12 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Mon Feb 4 20:36:49 2008 Subject: Post Translational Modification of Proteins In-Reply-To: References: Message-ID: <710764ea0802041500i451e69dev2dac59652a79c5a0@mail.gmail.com> On 04/02/2008, Sudheendra Rao N R wrote: > > Hi, > can somebody tell me what changes would occur to the molecular weight of a > protein being run in a denaturing PAGE after it undergoes Post > translational > modification? > like sumoylation can increase mol weight by 11kD. I don't know too much about it, however your question made me do a quick search for SUMO (small ubiquitin related modifiers) and here's a link that shows some antibodies that you could use for detecting the same (in this case, it is a kit that uses p53) and it seems plausible that the mol weight is modified by 11kDa. http://www.activemotif.com/catalog/protein_mod/sumolink best Pow Sudheendra. > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From akhan357 from sbcglobal.net Mon Feb 4 18:39:33 2008 From: akhan357 from sbcglobal.net (AK) Date: Mon Feb 4 20:36:54 2008 Subject: Post Translational Modification of Proteins References: Message-ID: Is this a trick question? :) "Sudheendra Rao N R" wrote in message news:mailman.491.1202159543.2451.methods@net.bio.net... > Hi, > can somebody tell me what changes would occur to the molecular weight of a > protein being run in a denaturing PAGE after it undergoes Post > translational > modification? > like sumoylation can increase mol weight by 11kD. > > Sudheendra. > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God From nobody from nospam.not Mon Feb 4 20:24:30 2008 From: nobody from nospam.not (Han) Date: Tue Feb 5 08:47:55 2008 Subject: How to prepare 1M H2O2 using 30% hydrogen peroxide? References: Message-ID: Aawara Chowdhury wrote in news:qHDpj.14164$M24.11783@newsfe17.lga: > In , > WANG XF wrote: > >> I want to use H2O2 as an inducer of apoptosis. How to prepare 1M H2O2 >> using 30% hydrogen peroxide? > > The molecular weight of H2O2 is 34 daltons (or close enough to 34). > 1M H2O2 = 34 g/L > 30% H2O2 = 300 g/L > > Do the math, and dilute it in ddH2O. > > AC Don't mouth-pipet the 30%. 3 % is OK, but don't overdo it - it tastes bad. -- Best regards Han email address is invalid From ucgatan from ucl.ac.uk Mon Feb 4 21:10:07 2008 From: ucgatan from ucl.ac.uk (Tom Anderson) Date: Tue Feb 5 08:48:05 2008 Subject: GFP, PBS and warmth In-Reply-To: References: Message-ID: On Sat, 2 Feb 2008, Tom Anderson wrote: > Can anyone tell me what my GFP-expressing cells will look like having > been fixed in formaldehyde and then incubated in PBS at 37 degrees for > twelve hours? Well, i found out: they're fine. The cells themselves look a bit unhappy, but i have a biological explanation for that, based on their being exposed to 240 volts of electric field twelve hours earlier. However ... > Note to self: in future, when doing a timecourse experiment, put the > coverslips for each timepoint in separate four-well dishes. There was another coverslip in another well, which was sat there in medium during fixation and washing of the first coverslip, and then waited another twelve hours before being fixed. Those cells are absolutely destroyed; collapsed, shrunken, warped, unholy travesties. My first thought on seeing them was to wonder how i was going to be able to burn glass, and where i was going to find a priest at this time of night. So, kids: formaldehyde is powerful stuff! Just having a 4% solution sitting near your cells for ten minutes is enough to do them over proper. So don't do that. tom -- Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT (t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson@ucl.ac.uk From wxfhome from gmail.com Tue Feb 5 09:45:08 2008 From: wxfhome from gmail.com (WANG XF) Date: Tue Feb 5 13:49:52 2008 Subject: How to prepare 1M H2O2 using 30% hydrogen peroxide? Message-ID: <404daa2f0802050645j41a4dba6g114dff21836e8714@mail.gmail.com> *Thanks for your help!* ** *30% H2O2 is 300g/L? or 300L/L or 300g/g?* ** *Some of my colleagues have different suggestions.* ** *Thank you!* ** *Lao WANG* ------------------------------ > > Message: 2 > Date: Mon, 4 Feb 2008 11:29:15 +0800 > From: "WANG XF" > Subject: How to prepare 1M H2O2 using 30% hydrogen peroxide? > To: methods@magpie.bio.indiana.edu > Message-ID: > <404daa2f0802031929ne677896q566a0ea84761e93c@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hello! > > I want to use H2O2 as an inducer of apoptosis. How to prepare 1M H2O2 > using > 30% hydrogen peroxide? > > Thank you all! > > Lao WANG > > > ------------------------------ > > Message: 3 > Date: Mon, 04 Feb 2008 15:49:22 +1100 > From: Bean Long > Subject: Re: How to prepare 1M H2O2 using 30% hydrogen peroxide? > To: methods@net.bio.net > Message-ID: <47a69952$1@clarion.carno.net.au> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed > > WANG XF wrote: > > Hello! > > > > I want to use H2O2 as an inducer of apoptosis. How to prepare 1M H2O2 > using > > 30% hydrogen peroxide? > > > > Thank you all! > > > > Lao WANG > > Here's a handy little link: > > http://www.trimen.pl/witek/calculators/stezenia.html > > > ------------------------------ > > Message: 4 > Date: Mon, 04 Feb 2008 12:38:46 GMT > From: Aawara Chowdhury > Subject: Re: How to prepare 1M H2O2 using 30% hydrogen peroxide? > To: methods@net.bio.net > Message-ID: > > In , > WANG XF wrote: > > > I want to use H2O2 as an inducer of apoptosis. How to prepare 1M H2O2 > using > > 30% hydrogen peroxide? > > The molecular weight of H2O2 is 34 daltons (or close enough to 34). > 1M H2O2 = 34 g/L > 30% H2O2 = 300 g/L > > Do the math, and dilute it in ddH2O. > > AC > -- > Email: echo 36434455860060025978157675027927670979097959886449930P | dc > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 33, Issue 3 > ************************************** > From novalidaddress from nurfuerspam.de Tue Feb 5 14:09:35 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Feb 5 21:15:42 2008 Subject: How to prepare 1M H2O2 using 30% hydrogen peroxide? References: Message-ID: <4f8737e1-bbe4-4542-8c1e-e98c7cddab5a@e6g2000prf.googlegroups.com> Dear Lao, that's the real issue. All your assumptions may be true. IMHO is is *absolutely* necessary to specify m/v (mass/volume), m/m or v/v. It even makes a difference if you (in this case rather hypothetically as solid H2O2 does not exist [at least for a long time]) mix 300 ml of H2O2 and 700ml of water, put 300 ml H2O2 in a beaker and add water up to 1liter or vice versa. Does not make always a real difference in the outcome of an experiment, but when you need to troubleshoot a protocol or reproduce an experiment, this kind of things will drive you nuts. There is nothing like a 'common sense' on how to make procentual solutions. If possible, always state molar contents, therefore. All the best, Wo On 5 Feb., 15:45, "WANG XF" wrote: > *Thanks for your help!* > ** > *30% H2O2 is 300g/L? or 300L/L or 300g/g?* > ** > *Some of my colleagues have different suggestions.* > ** > *Thank you!* > ** > *Lao WANG* > > ************************************** From yulia24 from gmail.com Tue Feb 5 15:26:14 2008 From: yulia24 from gmail.com (Yulia Shifrin) Date: Tue Feb 5 21:15:49 2008 Subject: Induction of apoptosis Message-ID: <9c7f9b9b0802051226j5f09338eyf6e8151a73437d7b@mail.gmail.com> Hi I've started doing some apoptosis studies recently, and it is a completely new field for me. I need a quick and easy way to induce apoptosis in 293 HEK cells as a positive control (I'm going to do annexin V staining on them). A time frame for my experiment will be only 4 hours, therefore I need a reagent that will make most of my cells annexin-positive fast. I've tried thapsigargin, but it works only after a prolonged exposure. I know that exposure to ethanol and peroxide may promote apoptosis, but i can't find a detailed protocol for that. Can you please offer a piece of advise for me? Thank you in advance. Yulia From bertrutten from gmail.com Wed Feb 6 06:10:42 2008 From: bertrutten from gmail.com (bertrutten@gmail.com) Date: Wed Feb 6 09:10:48 2008 Subject: buffer preparation Message-ID: <56cfe663-ccb7-4da3-a7ce-2a25962e06e5@i12g2000prf.googlegroups.com> Hello, How do i work this out? : I need 2mM H2O2 from a stock solution H2O2 of 30%, density 1.11 gr/ml, M.W. 34 anybody? thanks upfront b From nobody from nospam.not Wed Feb 6 06:45:38 2008 From: nobody from nospam.not (Han) Date: Wed Feb 6 09:10:54 2008 Subject: buffer preparation References: <56cfe663-ccb7-4da3-a7ce-2a25962e06e5@i12g2000prf.googlegroups.com> Message-ID: bertrutten@gmail.com wrote in news:56cfe663-ccb7-4da3-a7ce-2a25962e06e5 @i12g2000prf.googlegroups.com: > Hello, > > How do i work this out? >: > > I need 2mM H2O2 from a stock solution H2O2 of 30%, density 1.11 gr/ml, > M.W. 34 > > > anybody? > thanks upfront > > b > There is 3 problems here: Lack of understanding, lack of math knowledge and false precision. What is a 2 mM solution? Answer: A solution that contains 0.002 moles of the substance per liter. Or 2*34/1000 g H2O2 per liter. How much H2O2 is in a 30% solution with density 1.11? Answer: 30 g per 100 gram solution. Answer 2: volume of 100 gram solution with density 1.11: 100/1.11 ml (close enough). The rest of the math is up to the reader. False precision: 30% H2O2 does not stay like that forever. There is an expiration date. In addition, the 30% may always have been +/- 1-3%. 2 mM seems like a fairly low concentration to do much, but I'm not a redox maven. Please make sure you generate such solution freshly and with scrupulously clean glassware. Very small amounts of oxidizable contaminants will reduce the H2O2 content drastically. -- Best regards Han email address is invalid From piero.sestili from uniurb.it Wed Feb 6 10:02:26 2008 From: piero.sestili from uniurb.it (Prof. Piero Sestili) Date: Wed Feb 6 12:15:46 2008 Subject: R: buffer preparation In-Reply-To: <56cfe663-ccb7-4da3-a7ce-2a25962e06e5@i12g2000prf.googlegroups.com> Message-ID: <002501c868d1$4d9d7f00$7d0ca8c0@iram.it> First prepare a 2 M solution by diluting 2.26 ml of your stock to 10 ml, then make serial 1:10 dilutions down to 2 mM or prepare a 200 mM solution first diluting 226 ul of your stock to 10 ml and then another 1:100 dilution to 2 mM. As you prefer.... Piero Prof. Piero Sestili Istituto di Farmacologia e Farmacognosia e Centro di Ricerca sull'Attivit? Motoria Universit? degli Studi di Urbino "Carlo Bo" Via "I Maggetti" 26 61029 URBINO (PU) Tel. 0722 303414; 0722 305524 Fax 0722 303401 -----Messaggio originale----- Da: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] Per conto di bertrutten@gmail.com Inviato: mercoled? 6 febbraio 2008 12.11 A: methods@magpie.bio.indiana.edu Oggetto: buffer preparation Hello, How do i work this out? : I need 2mM H2O2 from a stock solution H2O2 of 30%, density 1.11 gr/ml, M.W. 34 anybody? thanks upfront b _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From pow.joshi from gmail.com Wed Feb 6 13:09:45 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Wed Feb 6 19:25:36 2008 Subject: How to prepare 1M H2O2 using 30% hydrogen peroxide? In-Reply-To: References: <4f8737e1-bbe4-4542-8c1e-e98c7cddab5a@e6g2000prf.googlegroups.com> Message-ID: <710764ea0802061009j76567393l72fa9680a3196de2@mail.gmail.com> On 05/02/2008, DK wrote: > > In article < > 4f8737e1-bbe4-4542-8c1e-e98c7cddab5a@e6g2000prf.googlegroups.com>, WS < > novalidaddress@nurfuerspam.de> wrote: > >Dear Lao, > > > >that's the real issue. All your assumptions may be true. > > > >IMHO is is *absolutely* necessary to specify m/v (mass/volume), m/m or > >v/v. It even makes a difference if you (in this case rather > >hypothetically as solid H2O2 does not exist [at least for a long > >time]) mix 300 ml of H2O2 and 700ml of water, put 300 ml H2O2 in a > >beaker and add water up to 1liter or vice versa. > > Industrial % concentrations tend to be m/m. This is clearly > the case, for example, with concentrated HCl (~ 37% ~ 12M) > or concentrated H3PO4 (85% = 15.2M). > > MSDS for 30% H2O2 says "balance water", suggesting > that indeed we are talking about 300 g + 700 g water. > In which case precise molarity calculattion become > troublesome even knowing density of 100% H2O2 > because water solutions can change volume > appreciably (e.g. 500 ml ethanol + 500 ml H2O > will result in less than 1000 ml solution). > > Not that any of that matters in the experiment in > question! Assuming no volume change and m/m > for 30% and density off 199% of 1.4 g/ml from > Wikipedia, we get 22-28% error depending on the > direction. With the "1 M" being clearly an arbitrary > round number, I wouln't worry about potential > 25% error as long as the way the solution is prepared > is clearly stated. yes, that is true.... H2O2 would be w/w.... the MSDS generally does'nt give the information, however, calling the company would give. Frankly, I did'nt know that this is true for all industrial preparation..... Thank you Dima. Pow DK > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From Antonio.Sarikas from gmail.com Wed Feb 6 14:39:05 2008 From: Antonio.Sarikas from gmail.com (AS) Date: Wed Feb 6 19:25:42 2008 Subject: Dulbecco`s PBS vs PBS pH7.4 Message-ID: <8dbed6dc-131a-4266-bfb8-e900ef4c575c@e23g2000prf.googlegroups.com> What is the difference between Dulbecco`s PBS vs PBS pH7.4? Does it make a difference if I just use it to wash cells (293, MEFs etc) before trypsination or lysation? Thanks! From metugenetics from yahoo.com Wed Feb 6 16:45:51 2008 From: metugenetics from yahoo.com (Ozan Aygun) Date: Wed Feb 6 19:25:51 2008 Subject: Induction of apoptosis Message-ID: <16475.93161.qm@web90402.mail.mud.yahoo.com> Hi, You may want to give try alpha-amanitin, which is a highly specific inhibitor of RNA polymerase II. It is known that exposure to this reagent causes apoptosis in human cells. Good luck, Ozan ____________________________________________________________________________________ Looking for last minute shopping deals? Find them fast with Yahoo! Search. http://tools.search.yahoo.com/newsearch/category.php?category=shopping From L.Kamalian from liverpool.ac.uk Thu Feb 7 07:39:37 2008 From: L.Kamalian from liverpool.ac.uk (Kamalian, Laleh) Date: Thu Feb 7 13:05:59 2008 Subject: Can you heat RPMI 1640? References: Message-ID: <31DC2DFFD70C174B8E3072803F894F91046C5E@EVSSTAFF1.livad.liv.ac.uk> Hi Thanks to everyone who answered my question regarding stable transfection of suspension cells. As DK had suggested I found ready made semi viscous media for suspension cells and bought it. However my cells are not growing in it, probably because the basic media is not the media that my cells are used to (RPMI 1640). I had suspected this before using it but wanted to give it a try. Now I want to make this using methylcellulose in my media. I have found the concentration of the components and every thing else. The only problem is how to dissolve methylcellulose in the media. I found out that I have to add warm (60 ' C) liquid to the powder and let it cool down while stirring. But can I heat RPMI 1640 media to 60"? I couldn't find the answer on the net. Thanks Laleh ________________________________ From: methods-bounces@oat.bio.indiana.edu on behalf of DK Sent: Sat 19/01/2008 22:38 To: methods@magpie.bio.indiana.edu Subject: Re: Stable transfection in suspension cells In article , "Kamalian, Laleh" wrote: >Hi to everyone. I am a PhD student and new to this mail list. I have a >problem I would like to ask. I am working with cells which grow in >suspension. I have managed to get a good nock down in transient >transfection using a plasmid vector and Lipofectamine as the transfection >reagent. Now I am ready to perform the stable transfection. However I >haven't got a clue how I will be able to separate the dead cells from >the live ones in the selection process especially after the massive death >happens. Furthermore, I don't know how I'm supposed to pick a single >clone even if I manage to get the live ones. No one in our lab has ever >transfected cells in suspension. The selection method I will use is >antibody selection (G418) for which I have already started the optimization >experiment in order to obtain the killing curve. I was wondering if any of >you have worked with suspension cells. Could you please give me some >help? Thanks >Laleh. This problem isn't new and there are several solutions. 1. Plate and select cells in 24-96 well format. For each well that gives you survivors and massive cell growth, purify one individual clone by limited dilution. (Google it). 2. Grow cells in viscous medium, so that clones stay together and, more or less, stay apart from each other. For mammalian cells a classic way to do that is carboxymethylcellulose mixed into medium. (Can't remember concentration but you should be able to google it - it's a standard thing in viral research). Then you just pick a clone with pipet tip under the miscoscope, expand and, to be sure it's clonal, purify by limited dilution. 3. If your cells survive overlay with a low-melting agarose, that's a convenient option. Overlay with medium containing G418 and 1% LM agarose 24-48 hours posttransfection. Once the clone is big enough, you just draw a circle around it and pick it with a tip or Paster pipette. This is routinely done for insect cells/baculovirus. The only drawback is that you need 2X concentrated medium since, obviously, you don't want to autoclave your medium. DK _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From tedesco from ibg.colorado.edu Thu Feb 7 17:17:13 2008 From: tedesco from ibg.colorado.edu (Pat Tedesco) Date: Thu Feb 7 18:00:48 2008 Subject: DNA alignment Message-ID: Did anyone find a program for PC's similar to DNA strider? Thanks! Pat Pat Tedesco Institute of Behavioral Genetics 1480 30th St. Boulder, CO 80303 303-492-2929 303-492-8063 FAX From strugd from ucla.edu Thu Feb 7 20:58:31 2008 From: strugd from ucla.edu (David Strugatsky) Date: Thu Feb 7 23:33:47 2008 Subject: DNA alignment References: Message-ID: On Thu, 07 Feb 2008 15:17:13 -0700, Pat Tedesco wrote: > Did anyone find a program for PC's similar to DNA strider? Thanks! Pat > > Pat Tedesco > Institute of Behavioral Genetics > 1480 30th St. > Boulder, CO 80303 > 303-492-2929 > 303-492-8063 FAX Try 'Ape' - A Plasmid Editor from http://www.biology.utah.edu/jorgensen/ wayned/ape/ Ape is similar to strider but far more advanced. It has many useful features to work with plasmid sequence and it also reads/writes strider and genbank formats. David David Strugatsky Postdoctoral Fellow David Geffen School of Medicine at UCLA Membrane Biology Laboratory/WLA VAMC 11301 Wilshire Blvd, Bldg 113, Rm 324 Los Angeles, CA 90073 From sudhee26 from gmail.com Thu Feb 7 23:10:28 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu Feb 7 23:33:56 2008 Subject: DNA alignment In-Reply-To: References: Message-ID: Try, 1. Gentle 2. Invitrogen-Vector NTI 3. DNA User 4. PlasmaDNA Sudheendra. On Feb 8, 2008 3:47 AM, Pat Tedesco wrote: > Did anyone find a program for PC's similar to DNA strider? Thanks! Pat > > Pat Tedesco > Institute of Behavioral Genetics > 1480 30th St. > Boulder, CO 80303 > 303-492-2929 > 303-492-8063 FAX > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From timegg113 from gmail.com Thu Feb 7 21:58:59 2008 From: timegg113 from gmail.com (Tim Eggleston) Date: Thu Feb 7 23:34:19 2008 Subject: Media left at 37 C O/N? Message-ID: The question regarding heating the RPMI media to 60 C brought to mind a question I've wondered about from time to time. If media, such as DMEM or MEM, is left in a 37 C water bath O/N, will be ok to use it the next day. I have always advised people to pitch it due to the possible degradation of L-glutamine, but I have always wondered how much degradation actually occurs, if any. Thanks! -- Tim Eggleston Horne Lab Research Assistant 2-505 Bowen Science Building University of Iowa From chemophoto from gmail.com Fri Feb 8 01:09:32 2008 From: chemophoto from gmail.com (chemophoto@gmail.com) Date: Fri Feb 8 13:32:03 2008 Subject: problems with restriction digestion Message-ID: <8110b835-31db-4e3e-a4cb-14fd1293c8b2@d4g2000prg.googlegroups.com> Hi all, Would really appreciate some help!! I am trying to clone a gene and this is giving me a lot of problems. When I started with an alkaline lysis prep of the plasmid and did a restriction digestion with BamHI and XbaI I was getting extra bands that should not be there. I than did a Midi prep of the plasmid and did two sequential digestions with BamHI and XbaI and my problem disappeared. I then went ahead with my cloning and finally got some transformants. I did an alkaline lysis prep of my transformants for screening and did some restriction digestions and it seems I have a few clones with the right profile. So I went ahead and did a Midi prep (to get a pure prep) to electroporate into my bug. After I did my midi prep I did a digestion with BamHI and XbaI (sequential) and again I am seeing extra bands that should not be there (the same bands I saw before). I used a 20ul digest, 1ul restriction enzyme, 2ul buffer and 1ul of plasmid DNA. I do not know the concentration of my plasmid DNA (didn't nanodrop it yet). I used 1ul plasmid DNA based on the agarose gel result of the midi prep. I digested it for 2hrs at 37 and did an EtOH pption and did the second digest. I am wondering why I am getting extra bands. Am I using too much enzyme? But I have used this amount of enzyme in digestions of 20ul before and it worked. Is there something else that I am doing wrong?? I thought the reason why I got the extra bands before was as I was using an alkaline lysis prep..which might have contaminants (RNA etc). Thanks a lot Mary31 From virashkgupta from gmail.com Fri Feb 8 08:15:31 2008 From: virashkgupta from gmail.com (Virash Gupta) Date: Fri Feb 8 13:32:49 2008 Subject: How to prepare 1M H2O2 using 30% hydrogen peroxide? Message-ID: 30 % H2O2 is 8.84 M solution. Dilute 8.84 times. The problem is solved. all the best On Feb 7, 2008 10:34 PM, wrote: > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. R: buffer preparation (Prof. Piero Sestili) > 2. Dulbecco`s PBS vs PBS pH7.4 (AS) > 3. Re: How to prepare 1M H2O2 using 30% hydrogen peroxide? > (Pow Joshi) > 4. RE:Induction of apoptosis (Ozan Aygun) > 5. Re: Dulbecco`s PBS vs PBS pH7.4 (DK) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 6 Feb 2008 16:02:26 +0100 > From: "Prof. Piero Sestili" > Subject: R: buffer preparation > To: , > Message-ID: <002501c868d1$4d9d7f00$7d0ca8c0@iram.it> > Content-Type: text/plain; charset="iso-8859-1" > > First prepare a 2 M solution by diluting 2.26 ml of your stock to 10 ml, > then make serial 1:10 dilutions down to 2 mM > or > prepare a 200 mM solution first diluting 226 ul of your stock to 10 ml > and then another 1:100 dilution to 2 mM. As you prefer.... > > > Piero > > > Prof. Piero Sestili > Istituto di Farmacologia e Farmacognosia e > Centro di Ricerca sull'Attivit? Motoria > Universit? degli Studi di Urbino "Carlo Bo" > Via "I Maggetti" 26 > 61029 URBINO (PU) > Tel. 0722 303414; 0722 305524 > Fax 0722 303401 > > > -----Messaggio originale----- > Da: methods-bounces@oat.bio.indiana.edu > [mailto:methods-bounces@oat.bio.indiana.edu] Per conto di > bertrutten@gmail.com > Inviato: mercoled? 6 febbraio 2008 12.11 > A: methods@magpie.bio.indiana.edu > Oggetto: buffer preparation > > Hello, > > How do i work this out? > : > > I need 2mM H2O2 from a stock solution H2O2 of 30%, density 1.11 gr/ml, > M.W. 34 > > > anybody? > thanks upfront > > b > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > > > > ------------------------------ > > Message: 2 > Date: Wed, 6 Feb 2008 11:39:05 -0800 (PST) > From: AS > Subject: Dulbecco`s PBS vs PBS pH7.4 > To: methods@net.bio.net > Message-ID: > <8dbed6dc-131a-4266-bfb8-e900ef4c575c@e23g2000prf.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > What is the difference between Dulbecco`s PBS vs PBS pH7.4? > > Does it make a difference if I just use it to wash cells (293, MEFs > etc) before trypsination or lysation? > > Thanks! > > > ------------------------------ > > Message: 3 > Date: Wed, 6 Feb 2008 13:09:45 -0500 > From: "Pow Joshi" > Subject: Re: How to prepare 1M H2O2 using 30% hydrogen peroxide? > To: DK > Cc: methods@magpie.bio.indiana.edu > Message-ID: > <710764ea0802061009j76567393l72fa9680a3196de2@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > On 05/02/2008, DK wrote: > > > > In article < > > 4f8737e1-bbe4-4542-8c1e-e98c7cddab5a@e6g2000prf.googlegroups.com>, WS < > > novalidaddress@nurfuerspam.de> wrote: > > >Dear Lao, > > > > > >that's the real issue. All your assumptions may be true. > > > > > >IMHO is is *absolutely* necessary to specify m/v (mass/volume), m/m or > > >v/v. It even makes a difference if you (in this case rather > > >hypothetically as solid H2O2 does not exist [at least for a long > > >time]) mix 300 ml of H2O2 and 700ml of water, put 300 ml H2O2 in a > > >beaker and add water up to 1liter or vice versa. > > > > Industrial % concentrations tend to be m/m. This is clearly > > the case, for example, with concentrated HCl (~ 37% ~ 12M) > > or concentrated H3PO4 (85% = 15.2M). > > > > MSDS for 30% H2O2 says "balance water", suggesting > > that indeed we are talking about 300 g + 700 g water. > > In which case precise molarity calculattion become > > troublesome even knowing density of 100% H2O2 > > because water solutions can change volume > > appreciably (e.g. 500 ml ethanol + 500 ml H2O > > will result in less than 1000 ml solution). > > > > Not that any of that matters in the experiment in > > question! Assuming no volume change and m/m > > for 30% and density off 199% of 1.4 g/ml from > > Wikipedia, we get 22-28% error depending on the > > direction. With the "1 M" being clearly an arbitrary > > round number, I wouln't worry about potential > > 25% error as long as the way the solution is prepared > > is clearly stated. > > > > yes, that is true.... H2O2 would be w/w.... the MSDS generally does'nt > give > the information, however, calling the company would give. Frankly, I > did'nt > know that this is true for all industrial preparation..... Thank you Dima. > > Pow > > DK > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > ------------------------------ > > Message: 4 > Date: Wed, 6 Feb 2008 13:45:51 -0800 (PST) > From: Ozan Aygun > Subject: RE:Induction of apoptosis > To: methods@magpie.bio.indiana.edu > Message-ID: <16475.93161.qm@web90402.mail.mud.yahoo.com> > Content-Type: text/plain; charset=iso-8859-1 > > Hi, > > You may want to give try alpha-amanitin, which is a > highly specific inhibitor of RNA polymerase II. It is > known that exposure to this reagent causes apoptosis > in human cells. > > Good luck, > > Ozan > > > > ____________________________________________________________________________________ > Looking for last minute shopping deals? > Find them fast with Yahoo! Search. > http://tools.search.yahoo.com/newsearch/category.php?category=shopping > > > > ------------------------------ > > Message: 5 > Date: Wed, 06 Feb 2008 22:25:02 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: Dulbecco`s PBS vs PBS pH7.4 > To: methods@net.bio.net > Message-ID: <1tqqj.50$iB4.41@newsfe07.lga> > > In article < > 8dbed6dc-131a-4266-bfb8-e900ef4c575c@e23g2000prf.googlegroups.com>, AS < > Antonio.Sarikas@gmail.com> wrote: > >What is the difference between Dulbecco`s PBS vs PBS pH7.4? > > Probably none. Gibco used to specify its Dulbecco's PBS > PH as 7.0-7.5. "PBS" is a mess. It can refer to a number of > similar solutions, each with and without Ca2+ and/or Mg2+. > > >Does it make a difference if I just use it to wash cells (293, MEFs > >etc) before trypsination or lysation? > > In the range in question, pH won't make a difference but for > trypsinization there might be a diffrence between PBS > containing divalents or not. Don't think it will be significant > if you use trypsin-EDTA. > > DK > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 33, Issue 6 > ************************************** > -- Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 From taskan4 from gmail.com Fri Feb 8 08:40:16 2008 From: taskan4 from gmail.com (TR) Date: Fri Feb 8 13:32:56 2008 Subject: Expression of a 8-kDa protein in E coli Message-ID: <70272d730802080540y541ff2a0j45616b073be0695b@mail.gmail.com> Hallo everybody, We are trying to express in E. coli a small protein (77 aa, 8090 Da), using the pRSET system and BL21 pLysS. We can't see any band of the expected size in our PAGEs (20% acrylamide), but we fear that such a small protein will disappear from the gel during staining (with colloidal coomassie). We are trying a different staining procedure in which small proteins are fixed before staining, described in this group a long time ago (see below). Any other suggestion to be able to 'see' this small recombinant protein? Thank you in advance, CG Quoted from Frank O. Fackelmayer (Fri Dec 7 10:20:53 EST 2001): >> in many cases, small proteins are not fixed well in the gel, and readily >> diffuse out during the staining process. We had the same problem, until >> we switched to a different formulation of the coomassie staining >> solution. Using one with formaldehyde provides a much better fixation, >> and a much higher sensitivity for small proteins. The formulation is >> described in Steck et al, 1980, Anal. Biochem. 107:21-24. >> >> Staining solution: >> 18ml EtOH, 42ml Water, 10ml formaldehyde (from 37% stock), 0.08g Coomassie >> (as always with coomassie solutions, do NOT reuse!) >> >> destaining solution: >> 250ml EtOH, 750ml water, 10ml formaldehyde >> >> staining takes approximately the double time, we used 1h for minigels. >> Replace with destaining solution as usual, until bands are well visible. >> In contrast to normal coomassie staining procedures, the sensitivity >> increases quite significantly when you store the gel in destaining >> solution plus 1/10 volume staining solution, overnight in the fridge. From jg374 from mole.bioc.cam.ac.uk Fri Feb 8 11:25:21 2008 From: jg374 from mole.bioc.cam.ac.uk (James Giddings) Date: Fri Feb 8 13:33:01 2008 Subject: Media left at 37 C O/N? In-Reply-To: References: Message-ID: Tim Eggleston wrote: > The question regarding heating the RPMI media to 60 C brought to mind > a question I've wondered about from time to time. If media, such as > DMEM or MEM, is left in a 37 C water bath O/N, will be ok to use it > the next day. I have always advised people to pitch it due to the > possible degradation of L-glutamine, but I have always wondered how > much degradation actually occurs, if any. Thanks! > IIRC Glutamine at 37 has a "half-life" of about 1 week, and 2 weeks at RT. Though other factors may affect the media's suitability for use such as how any ammonium ions from glutamine breakdown are buffered by other components. From novalidaddress from nurfuerspam.de Fri Feb 8 17:00:16 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Fri Feb 8 18:54:57 2008 Subject: problems with restriction digestion References: <8110b835-31db-4e3e-a4cb-14fd1293c8b2@d4g2000prg.googlegroups.com> Message-ID: <1b809df0-66ac-47ce-9707-03466955e5ac@f10g2000hsf.googlegroups.com> Hi Mary, can you run your DNA preps on the gel, too?. A too long alkakline step (some ptotocols say 5min) definitely will kill your plamsid (partially) and cause it to wind up in some strange undigestable conformation that may appear as extra band. For standards E.coli lab strains, a few seconds definitely are enough and you should work on ice and immediately proceed with neutralization. Beware, too, of XbaI sensitivity to certain methylation patterns possibly causing poor or partial digests. Best regards, Wo From novalidaddress from nurfuerspam.de Fri Feb 8 17:08:08 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Fri Feb 8 18:55:08 2008 Subject: Permanent labelling of -150degC metal racks? Message-ID: <7029f75d-2e6c-4ead-8986-ca30d9211f58@s13g2000prd.googlegroups.com> Dear experts, what would you recommend to label metal racks for -150 degC freezers? I want to write some letters (A,B,C, ..) on them in a way that it won't go off after a few weeks and also is readible when covered with a little ice. I thought of using an oridinary water proof pen, but maybe there is something better that you might want to recommend. Actually, attaching fluorescent plates (the colder the fluo...) with rivets would be nice, but I have no idea where to get such plastic?plates from and which material will survive these somwhat extreme temperatures and possible temperature changes. Any ideas? Wo From sudhee26 from gmail.com Fri Feb 8 18:24:59 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Fri Feb 8 18:55:13 2008 Subject: problems with restriction digestion In-Reply-To: <8110b835-31db-4e3e-a4cb-14fd1293c8b2@d4g2000prg.googlegroups.com> References: <8110b835-31db-4e3e-a4cb-14fd1293c8b2@d4g2000prg.googlegroups.com> Message-ID: Hi, Just to clarify! How have to come to that conclusion that the bands u are seeing are extra? sometimes insert might have restriction enzyme sites (i guess u are using two enzymes on either side of MCS). Try putting your insert virtually into the plasmid..using any plasmid manipulation software (Vector NTI, DNAuser, plasmaDNA, Gentle etc)..cut it with the enzymes you are about to digest it with (after circularizing it)..and see how many bands u get (neb cutter is also okay).. if you have done this job and still getting extra bands then 1. either your Enzymes are contaminated 2. of your mixture is receiving extra DNA from outside(check the digestion components..especially water) 3. something else!! sudheendra. On Feb 8, 2008 11:39 AM, wrote: > Hi all, > Would really appreciate some help!! > I am trying to clone a gene and this is giving me a lot of problems. > When I started with an alkaline lysis prep of the plasmid and did a > restriction digestion with BamHI and XbaI I was getting extra bands > that should not be there. I than did a Midi prep of the plasmid and > did two sequential digestions with BamHI and XbaI and my problem > disappeared. I then went ahead with my cloning and finally got some > transformants. I did an alkaline lysis prep of my transformants for > screening and did some restriction digestions and it seems I have a > few clones with the right profile. > So I went ahead and did a Midi prep (to get a pure prep) to > electroporate into my bug. After I did my midi prep I did a digestion > with BamHI and XbaI (sequential) and again I am seeing extra bands > that should not be there (the same bands I saw before). > > I used a 20ul digest, 1ul restriction enzyme, 2ul buffer and 1ul of > plasmid DNA. I do not know the concentration of my plasmid DNA (didn't > nanodrop it yet). I used 1ul plasmid DNA based on the agarose gel > result of the midi prep. I digested it for 2hrs at 37 and did an EtOH > pption and did the second digest. > > I am wondering why I am getting extra bands. Am I using too much > enzyme? But I have used this amount of enzyme in digestions of 20ul > before and it worked. Is there something else that I am doing > wrong?? > I thought the reason why I got the extra bands before was as I was > using an alkaline lysis prep..which might have contaminants (RNA > etc). > > Thanks a lot > Mary31 > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From w.ingram from uq.edu.au Fri Feb 8 20:14:41 2008 From: w.ingram from uq.edu.au (Wendy Ingram) Date: Sat Feb 9 16:12:29 2008 Subject: Permanent labelling of -150degC metal racks? Message-ID: <47ACFE81.1040104@uq.edu.au> Hi Wo, You might like to try what we do here - "dog tags" attached with a short length of brass bead chain to the handle of each rack. I haven't tried these at -150, but have used them for a long time in -80 freezers so I don't think there would be any problem. Our tags are about 3 cm diameter circles made of brass and engraved with a big black letter or number. The great thing with these is they are easy (and reasonably cheap) to get as they are used heaps in heavy industry for permanent labelling of pipes, valves etc. You can buy sets already pre-engraved (e.g A to Z or 1 to 20, 1 to 100 etc) from industrial safety shops, and many can also do custom engraving of blanks. We get ours from Seton (they are a big company in USA and Australia etc, no affiliation), where they are called "brass valve tags". Very easy to see through frost and ice, plus easy to wipe. In my experience they go forever. Cheers, Wendy. -- Wendy Ingram, PhD Senior Research Officer RCH Cancer Research Laboratory Phone: +61 7 3636-9211 Fax: +61 7 3365-5455 Email: w.ingram atatat uq.edu.au Postal & Delivery Address: Department of Paediatrics and Child Health The University of Queensland Level 3, Foundation Building Royal Children's Hospital Herston Rd, Herston, QLD 4029 Australia From athelas0113 from gmail.com Fri Feb 8 21:17:04 2008 From: athelas0113 from gmail.com (Z.L. K) Date: Sat Feb 9 16:12:36 2008 Subject: question about phenol/water saturated solution Message-ID: <900088c20802081817hb37a8d9r550a03d966fce448@mail.gmail.com> Hi all, I have a question with regard to the phenol/water saturated solution. I didn't add any acids into it when i made it, when i test the pH of the aqueous phase it said below 5 with the pH paper. the phenol was a fresh one from some commercial company back then. currently the pH of the aqueous phase is still the same after half a year sitting in the cold room at 4 C. Today my labmate brought up a question for me and asked me what acid I added into to to bring down the phenol solution pH. I told him I didn't add anything. based on my understanding from reading online literatures before, phenol is an organic acid and its pH stays low unless you use some buffer to bring it up to the point you want it to be (such as pH 7 for extracting DNA). I wonder if my understanding is correct or not, as i couldn't find any detail online information now, which is weird. My question here then should include two parts: 1, i have had RNA extracted with this phenol/water saturated buffer (well then it's from the in vitro transcription reaction, quite pure an RNA yield anyway), does it mean that the water-saturated phenol really has the right pH to extract RNA (just to answer my labmate's question); 2, if not, what acid should be used in adjusting the pH? Thanks! z. From pooja from mbu.iisc.ernet.in Sat Feb 9 06:54:40 2008 From: pooja from mbu.iisc.ernet.in (pooja@mbu.iisc.ernet.in) Date: Sat Feb 9 16:12:49 2008 Subject: Enterohaemorrhagic Escherichia coli Message-ID: <1705.10.128.17.11.1202558080.squirrel@mbu.iisc.ernet.in> Dear Sir, I am Pooja, a student at IISc, India. I was reading about pathogenic E.coli and just had a simple query. Sir, do you need any special facility to work with pathogenic E.coli like 0155:H7 or can we work in the normal hood used for culturing nonpathogenic E.coli. Are any specific precautions required while working with pathogenic E.coli? Waiting eagerly for your reply. Regards, Pooja -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. From mlsulliv from wisc.edu Sat Feb 9 10:16:04 2008 From: mlsulliv from wisc.edu (Michael Sullivan) Date: Sat Feb 9 16:12:54 2008 Subject: problems with restriction digestion In-Reply-To: <8110b835-31db-4e3e-a4cb-14fd1293c8b2@d4g2000prg.googlegroups.com> References: <8110b835-31db-4e3e-a4cb-14fd1293c8b2@d4g2000prg.googlegroups.com> Message-ID: <6A6CEB7D-E0D1-4FF3-94CA-1C0157AEB529@wisc.edu> Another thing to keep in mind is how you incubate the digestion reaction. Lots of people incubate digestioin reactions in a waterbath. Be very careful doing this, especially with enzymes that have star activity. In a waterbath, what often happens is that the bottom of the tube is warm, but the top of the tube is cool. Water evaporates from the reaction and condenses on the top, increasing the concentration of enzyme, salt and glycerol in the digest. For small volume digests this effect can be significant, and I've certainly seen BamHI digestions exhibit star activity when incubated in a waterbath. I prefer to carry out my digest incubations in a warm air incubator: initial heat transfer to the reaction is slower, but there is no problem with evaporation/condensation since warm air surrounds the entire tube. Mike On Feb 8, 2008, at 12:09 AM, chemophoto@gmail.com wrote: > > > I used a 20ul digest, 1ul restriction enzyme, 2ul buffer and 1ul of > plasmid DNA. I do not know the concentration of my plasmid DNA (didn't > nanodrop it yet). I used 1ul plasmid DNA based on the agarose gel > result of the midi prep. I digested it for 2hrs at 37 and did an EtOH > pption and did the second digest. > > I am wondering why I am getting extra bands. Am I using too much > enzyme? But I have used this amount of enzyme in digestions of 20ul > before and it worked. Is there something else that I am doing > wrong?? > I thought the reason why I got the extra bands before was as I was > using an alkaline lysis prep..which might have contaminants (RNA > etc). > > --- Michael L. Sullivan Plant Research Molecular Geneticist US Dairy Forage Research Center ARS-USDA 1925 Linden Drive West Madison, WI 53706 (608) 890-0046 (Phone) (608) 890-0076 (FAX) From novalidaddress from nurfuerspam.de Sat Feb 9 17:25:42 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Sat Feb 9 20:52:38 2008 Subject: Enterohaemorrhagic Escherichia coli References: Message-ID: Dear Pooja, that mostly depends on you local governmental regulations. In general, the best thing to do for you is to ask the people in charge for biosafety issues at your facility. Here in Germany, it would be at least biosafety lavel 2 (L2) for work with whole pathogenic bacteria and genetic engineering safety lavel 2, too (S2) if you work with single genes from such an organism. If you plan to investigate with factors that influence virulence, the safety standards to follow will be higher than if you just want to do genotyping by sequencing some probably not dangerous parts of their chromosomes in order to characterize isolates from the clinic. Another issue might be that you don't want to contaminate any standard lab cultures of E. coli with this probably quite nasty bug. Not to imagine what will happen if people start to grow their DNA preps in hemorragic E. coli strains, not being aware of what is lurking inside their flasks. Getting in contact with large amounts of 'your' strain probably will cause serious health issues to everyone affected. And in the end to you: When they find out where the bug came from, they will chase you to the South Pole or to Mars and Venus :-). So you probably want to work in well separated labs and also have all necessary precautions for efficient decontamination of all used labware and disposals containing your bacteria. Have fun, Wo From sudhee26 from gmail.com Sun Feb 10 00:06:50 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Sun Feb 10 00:30:39 2008 Subject: Enterohaemorrhagic Escherichia coli In-Reply-To: References: Message-ID: Hi Pooja, Under DBT guidelines all Entero Pathogenic strains of E Coli come under Risk Category II http://dbtbiosafety.nic.in/act/_Annex-4.htm Try mailing them or contacting a local wing of that. Sudheendra. On Feb 10, 2008 10:36 AM, Sudheendra Rao N R wrote: > > > On Feb 10, 2008 3:55 AM, WS wrote: > > > Dear Pooja, > > > > that mostly depends on you local governmental regulations. In general, > > the best thing to do for you is to ask the people in charge for > > biosafety issues at your facility. > > > > Here in Germany, it would be at least biosafety lavel 2 (L2) for work > > with whole pathogenic bacteria and genetic engineering safety lavel 2, > > too (S2) if you work with single genes from such an organism. > > > > If you plan to investigate with factors that influence virulence, the > > safety standards to follow will be higher than if you just want to do > > genotyping by sequencing some probably not dangerous parts of their > > chromosomes in order to characterize isolates from the clinic. > > > > Another issue might be that you don't want to contaminate any standard > > lab cultures of E. coli with this probably quite nasty bug. Not to > > imagine what will happen if people start to grow their DNA preps in > > hemorragic E. coli strains, not being aware of what is lurking inside > > their flasks. Getting in contact with large amounts of 'your' strain > > probably will cause serious health issues to everyone affected. And in > > the end to you: When they find out where the bug came from, they will > > chase you to the South Pole or to Mars and Venus :-). So you probably > > want to work in well separated labs and also have all necessary > > precautions for efficient decontamination of all used labware and > > disposals containing your bacteria. > > > > Have fun, > > > > Wo > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God -- Think before agree Think before you nod but STOP thinking and You Are God From zineldeen from gmail.com Sat Feb 9 23:25:44 2008 From: zineldeen from gmail.com (doaa zineldeen) Date: Sun Feb 10 00:30:44 2008 Subject: site directed mutagensis Message-ID: <440923e10802092025t5baa04dvb94a2cbcb33db9cf@mail.gmail.com> hello I just want if anyone have a protocol for pcr mediated site directed mutagensis without using stratagene kit i have a template vector containing the insert and i want to mutate k81 into A i designed 2 primers flanking the mutation site and i want can i perform 2 pcr steps and a third mix with normal polymerase i am using phusion can i proceed to get the new product containing the mutated sites If someone can help i am really grateful Doaa From kaj.stenberg from helsinki.fi.invalid Sun Feb 10 06:08:15 2008 From: kaj.stenberg from helsinki.fi.invalid (kaj.stenberg@helsinki.fi.invalid) Date: Sun Feb 10 14:22:29 2008 Subject: Kd semantics Message-ID: This is probably trivial, but it confuces me: If the Kd for a ligand is mM it binds poorly, if the Kd is nM it binds well. But in which case does one refer to the Kd as being "higher" or "lower"? -- Kaj From DSpencer from NO!!SPAM.Dal.CA Sun Feb 10 20:05:37 2008 From: DSpencer from NO!!SPAM.Dal.CA (David F. Spencer) Date: Mon Feb 11 00:02:29 2008 Subject: question about phenol/water saturated solution In-Reply-To: References: Message-ID: <47af9f63$0$4066$9a566e8b@news.aliant.net> Z.L. K wrote: > Hi all, > I have a question with regard to the phenol/water saturated solution. I > didn't add any acids into it when i made it, when i test the pH of the > aqueous phase it said below 5 with the pH paper. the phenol was a fresh one > from some commercial company back then. currently the pH of the aqueous > phase is still the same after half a year sitting in the cold room at 4 C. > Today my labmate brought up a question for me and asked me what acid I added > into to to bring down the phenol solution pH. I told him I didn't add > anything. based on my understanding from reading online literatures before, > phenol is an organic acid and its pH stays low unless you use some buffer to > bring it up to the point you want it to be (such as pH 7 for extracting > DNA). I wonder if my understanding is correct or not, as i couldn't find any > detail online information now, which is weird. > > My question here then should include two parts: 1, i have had RNA extracted > with this phenol/water saturated buffer (well then it's from the in vitro > transcription reaction, quite pure an RNA yield anyway), does it mean that > the water-saturated phenol really has the right pH to extract RNA (just to > answer my labmate's question); 2, if not, what acid should be used in > adjusting the pH? > > Thanks! > > z. The classic name for phenol is carbolic acid and it is indeed a very weak acid, pka of a little under 10, and pH of a water solution of about 6 or less. I wouldn't trust pH paper with phenol but instead would use a pH meter. For protocols where RNA is desired but not DNA, the usual strategy is to have the solution contain an acetic acid/sodium acetate buffer (say 0.3M) at about pH 4.5, and use phenol equilibrated against a similar buffer. Your approach is essentially the same, although as with any method, using unbuffered solutions will occasionally lead to unpredictable results. David Spencer From L.Kamalian from liverpool.ac.uk Sun Feb 10 15:15:56 2008 From: L.Kamalian from liverpool.ac.uk (Kamalian, Laleh) Date: Mon Feb 11 00:02:36 2008 Subject: Media left at 37 C O/N? In-Reply-To: References: Message-ID: Hi We are usually advised not to leave the media in the water bath for long. However considering we grow our cells in the same media at 37' it shoudnt't really do much damage to the media. The only thing is that if you are using antibiotics in your media (like pen/strep) they degrade after 3 days at 37' and this is one of reasons we have to change the media after 2-3 days. Laleh From: Tim Eggleston Sent: Fri 08/02/2008 02:58 To: methods@magpie.bio.indiana.edu Subject: Media left at 37 C O/N? The question regarding heating the RPMI media to 60 C brought to mind a question I've wondered about from time to time. If media, such as DMEM or MEM, is left in a 37 C water bath O/N, will be ok to use it the next day. I have always advised people to pitch it due to the possible degradation of L-glutamine, but I have always wondered how much degradation actually occurs, if any. Thanks! -- Tim Eggleston Horne Lab Research Assistant 2-505 Bowen Science Building University of Iowa _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From kjaanson from gmail.com Sun Feb 10 15:56:37 2008 From: kjaanson from gmail.com (kjaanson@gmail.com) Date: Mon Feb 11 00:02:44 2008 Subject: CHEF gel genomic DNA restriction Message-ID: <2a40d57d-b1c2-4e97-b458-a13271d6d03e@i12g2000prf.googlegroups.com> Hi I have lately tried to restrict the genomic DNA of mammalian cells in agarose plugs and then separate the fragments with CHEF pulse field. The restriction seems to be working but the restricted DNA migrates weird in gel. It is very dense in the Kb-Mb range (EtBr staining shows that the DNA in that range has kind of spread over the lane borders). After the dense part ends the DNA occupies only the central part of the lane. Southern plot of the gel shows that the DNA runs higher (more slowly) than it should be, also the bands are a bit distorted. I was wondering if the anomaly might be associated with the concentration of DNA in agarose plug, to my calculations there should be about 3ug of genomic DNA in one plug. If that is the case then could I vary the pulse field parameters so that the high amount of genomic DNA would migrate normally? Kaur Jaanson From novalidaddress from nurfuerspam.de Sun Feb 10 16:08:50 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Mon Feb 11 00:02:50 2008 Subject: inducible rodent ecotropic retroviral vector for siRNA? Message-ID: Dear Experts, actually, all what I am looking for is in the subject. We want to express / inhbit a gene product which will among other effects, inhibit cell cycle progression and induce apoptosis. That's why it might be a good idea to suppress the expression of the introduced gene while the cell line is under constrcution. I thought of using a retroviral system (like pLNCX) for making stable cells (murine or rat myoblast and preadipocyte cell lines) first and then induce the gene expression/knockdown after the cells have been differentiated. Can you suggest any system that is likely to work for this purpose (low or no leakage before induction, good induction control of expression after induction, reasonable price of inducer - eg Tamoxifen or similar). Also, if someone knows of a siRNA vector (H1 or U6 promoter) that is sort of freely available ('GPL'), I'd be grateful for any hints. Many thanks, Wo hubahopp ata gmx dot de From novalidaddress from nurfuerspam.de Sun Feb 10 16:10:26 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Mon Feb 11 00:03:00 2008 Subject: CHEF gel genomic DNA restriction References: <2a40d57d-b1c2-4e97-b458-a13271d6d03e@i12g2000prf.googlegroups.com> Message-ID: <37b86d35-0dce-47ff-a595-7c2c213ddb5e@j20g2000hsi.googlegroups.com> Too much salt? Wo From kaj.stenberg from helsinki.fi.invalid Mon Feb 11 02:50:12 2008 From: kaj.stenberg from helsinki.fi.invalid (kaj.stenberg@helsinki.fi.invalid) Date: Mon Feb 11 13:43:57 2008 Subject: Kd semantics References: Message-ID: DK wrote: > In article , kaj.stenberg@helsinki.fi.invalid wrote: >>This is probably trivial, but it confuces me: >> >>If the Kd for a ligand is mM it binds poorly, if the Kd is nM it binds >>well. But in which case does one refer to the Kd as being "higher" or >>"lower"? > mM is "higher" than nM. However, sometimes people switch from > talking about Kd to talking about "affinity". Affinity is "higher" when > Kd ~ nM. Thank you, "affinity" is of course unambigious, I did just not come to think of it. Just another thing: when can you start talking about a "real" (as in specific) affinity? I am reading papers with a Kd in the ballpark of tens of millimolar and find it hard to repeat the work so that I truly believe in the results, since every random event seems to affect the curves fairly much. -- Kaj From joseolucha from gmail.com Mon Feb 11 10:34:55 2008 From: joseolucha from gmail.com (Jose Olucha) Date: Mon Feb 11 13:44:05 2008 Subject: Calcium binding protein assay Message-ID: First time I post something in the methods mail list (very exciting) Anywhoo... I am working with a CIB1 (calcium binding protein involved in cell development, migration and differentiation) ortholog(F30A10.1) in C. elegans. I was looking for any quick and easy calcium binding assays that I could use as a possible activity assay. Also question number 2: would anyone know of a reason why WB would not work using nitrocellulose as blotting membrane, as opposed to PVDF? Blots of this protein work with PVDF membrane but do not work with nitrocellulose. Any ideas why? From piero.sestili from uniurb.it Mon Feb 11 04:23:50 2008 From: piero.sestili from uniurb.it (Prof. Piero Sestili) Date: Mon Feb 11 13:49:41 2008 Subject: R: CHEF gel genomic DNA restriction(No virus check: scan engine not ready) In-Reply-To: <2a40d57d-b1c2-4e97-b458-a13271d6d03e@i12g2000prf.googlegroups.com> Message-ID: <001001c86c8f$d05e6730$7d0ca8c0@iram.it> Dear Kaur, post the running conditions you're using then we can talk about the CHEF protocol modifications. Cheers Prof. Piero Sestili Istituto di Farmacologia e Farmacognosia e Centro di Ricerca sull'Attivit? Motoria Universit? degli Studi di Urbino "Carlo Bo" Via "I Maggetti" 26 61029 URBINO (PU) Tel. 0722 303414; 0722 305524 Fax 0722 303401 -----Messaggio originale----- Da: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] Per conto di kjaanson@gmail.com Inviato: domenica 10 febbraio 2008 21.57 A: methods@magpie.bio.indiana.edu Oggetto: CHEF gel genomic DNA restriction(No virus check: scan engine not ready) Hi I have lately tried to restrict the genomic DNA of mammalian cells in agarose plugs and then separate the fragments with CHEF pulse field. The restriction seems to be working but the restricted DNA migrates weird in gel. It is very dense in the Kb-Mb range (EtBr staining shows that the DNA in that range has kind of spread over the lane borders). After the dense part ends the DNA occupies only the central part of the lane. Southern plot of the gel shows that the DNA runs higher (more slowly) than it should be, also the bands are a bit distorted. I was wondering if the anomaly might be associated with the concentration of DNA in agarose plug, to my calculations there should be about 3ug of genomic DNA in one plug. If that is the case then could I vary the pulse field parameters so that the high amount of genomic DNA would migrate normally? Kaur Jaanson _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From kjaanson from gmail.com Mon Feb 11 04:35:48 2008 From: kjaanson from gmail.com (Kaur Jaanson) Date: Mon Feb 11 13:49:47 2008 Subject: R: CHEF gel genomic DNA restriction(No virus check: scan engine not ready) In-Reply-To: <001001c86c8f$d05e6730$7d0ca8c0@iram.it> References: <2a40d57d-b1c2-4e97-b458-a13271d6d03e@i12g2000prf.googlegroups.com> <001001c86c8f$d05e6730$7d0ca8c0@iram.it> Message-ID: <4f6282d40802110135m267c881p8a83dc5777e3f01e@mail.gmail.com> Hi Sorry, forgot to add the conditions. I use 1.2% agarose gel with 0.5x TAE buffer, running parameters are 5.4 V/cm field strength, 2-90 sec switch time, 19 hr run time. Biorad CHEF DR-II system to run the pulse field electrophoresis. Kaur Jaanson On Feb 11, 2008 11:23 AM, Prof. Piero Sestili wrote: > Dear Kaur, > > post the running conditions you're using then we can talk about the CHEF > protocol modifications. > > Cheers > > Prof. Piero Sestili > Istituto di Farmacologia e Farmacognosia e > Centro di Ricerca sull'Attivit? Motoria > Universit? degli Studi di Urbino "Carlo Bo" > Via "I Maggetti" 26 > 61029 URBINO (PU) > Tel. 0722 303414; 0722 305524 > Fax 0722 303401 > > > -----Messaggio originale----- > Da: methods-bounces@oat.bio.indiana.edu > [mailto:methods-bounces@oat.bio.indiana.edu] Per conto di > kjaanson@gmail.com > Inviato: domenica 10 febbraio 2008 21.57 > A: methods@magpie.bio.indiana.edu > Oggetto: CHEF gel genomic DNA restriction(No virus check: scan engine > not ready) > > Hi > > I have lately tried to restrict the genomic DNA of mammalian cells in > agarose plugs and then separate the fragments with CHEF pulse field. > The restriction seems to be working but the restricted DNA migrates > weird in gel. It is very dense in the Kb-Mb range (EtBr staining shows > that the DNA in that range has kind of spread over the lane borders). > After the dense part ends the DNA occupies only the central part of > the lane. Southern plot of the gel shows that the DNA runs higher > (more slowly) than it should be, also the bands are a bit distorted. > > I was wondering if the anomaly might be associated with the > concentration of DNA in agarose plug, to my calculations there should > be about 3ug of genomic DNA in one plug. If that is the case then > could I vary the pulse field parameters so that the high amount of > genomic DNA would migrate normally? > > > Kaur Jaanson > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > -- Kaur Jaanson From scoop from mail.nih.gov Mon Feb 11 17:45:38 2008 From: scoop from mail.nih.gov (Sharon) Date: Mon Feb 11 20:46:28 2008 Subject: Problems doing Northern Blot with small probes (oligo, riboprobe, PCR and random primed) Message-ID: Dear All, I am having problems doing a Northern Blot with small probes. I am able to do a great Northern Blot with a random primed probe that is 500 bp or longer. However, when I try to do a blot with a 200 bp or shorter probe I get no signal. I am using radiolabeled probes. I have tried kinased oligo probes, Starfire probes (IDT), riboprobes, PCR'd probes and random primed probes. I wonder if the problem is that my message is rare and I don't get enough signal - maybe the random primed larger probe concatamerizes and increases signal that way, but I just don't know. I am using Expresshyb (clontech) or Ultrahyb (ambion) with no difference (no signal). When I decrease hybridization or washing temperature/stringency I still haven't gotten a signal (down to 37C with high stringency (2x SSC) wash buffer only. I can't imagine what I'm doing wrong (I expect it's something silly and obvious). I would appreciate any advice, questions, etc. Thanks in advance, Sharon From pjie2 from cam.ac.uk Tue Feb 12 03:44:26 2008 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Tue Feb 12 12:28:16 2008 Subject: Problems doing Northern Blot with small probes (oligo, riboprobe, PCR and random primed) References: Message-ID: <61d4jaF1u0kkcU1@mid.individual.net> Sharon wrote: > >I am having problems doing a Northern Blot with small probes. I am >able to do a great Northern Blot with a random primed probe that is >500 bp or longer. However, when I try to do a blot with a 200 bp or >shorter probe I get no signal. What are you using as your probe? Where do they map within the gene you're studying? Have you tried several different short sequences, or is it one particular sequence that's failing? It could (for example) be that your 200bp probe is derived from an alternatively spliced exon that's simply not expressed in the tissues you're investigating. Next, are you sure that the larger probe is detecting the right thing? Are there repeated sequences in the other 300bp that might cross-hybridize and detect some other transcript? Is the gene you're studying a singleton, or is it part of a larger gene family? Peter From mebewafa from yahoo.com Tue Feb 12 03:28:08 2008 From: mebewafa from yahoo.com (prabhat khadka) Date: Tue Feb 12 12:28:26 2008 Subject: loooking for protein phosphorylation method Message-ID: <451710.56469.qm@web51812.mail.re2.yahoo.com> hi If anyone could help me with protein phosphorylation protocal , taht will be highly appericated. hope to see soon ____________________________________________________________________________________ Looking for last minute shopping deals? Find them fast with Yahoo! Search. http://tools.search.yahoo.com/newsearch/category.php?category=shopping From kjaanson from gmail.com Tue Feb 12 03:13:05 2008 From: kjaanson from gmail.com (Kaur Jaanson) Date: Tue Feb 12 12:30:15 2008 Subject: R: CHEF gel genomic DNA restriction(No virus check: scan engine not ready) In-Reply-To: References: <2a40d57d-b1c2-4e97-b458-a13271d6d03e@i12g2000prf.googlegroups.com> <001001c86c8f$d05e6730$7d0ca8c0@iram.it> <4f6282d40802110135m267c881p8a83dc5777e3f01e@mail.gmail.com> Message-ID: <4f6282d40802120013u45deb600t2c6220aaf4d4c3cf@mail.gmail.com> Hi I have already read that manual, unfortunately it doesn't say anything about the DNA concentrations effect on the mobility. I am currently trying to test if the restriction enzyme buffer might cause the problem. On Feb 12, 2008 6:43 AM, Sudheendra Rao N R wrote: > Hey, > i found this one on net. > check if it can troubleshoot some of your problems. > I think looking again into your protocol would do lot of help. > > http://humgen.wustl.edu/hdk_lab_manual/12/12_2.html > > Sudheendra. > > > > On Feb 11, 2008 3:05 PM, Kaur Jaanson wrote: > > > Hi > > > > Sorry, forgot to add the conditions. > > > > I use 1.2% agarose gel with 0.5x TAE buffer, running parameters are > > 5.4 V/cm field strength, 2-90 sec switch time, 19 hr run time. > > Biorad CHEF DR-II system to run the pulse field electrophoresis. > > > > Kaur Jaanson > > > > > > > > > > On Feb 11, 2008 11:23 AM, Prof. Piero Sestili > wrote: > > > Dear Kaur, > > > > > > post the running conditions you're using then we can talk about the CHEF > > > protocol modifications. > > > > > > Cheers > > > > > > Prof. Piero Sestili > > > Istituto di Farmacologia e Farmacognosia e > > > Centro di Ricerca sull'Attivit? Motoria > > > Universit? degli Studi di Urbino "Carlo Bo" > > > Via "I Maggetti" 26 > > > 61029 URBINO (PU) > > > Tel. 0722 303414; 0722 305524 > > > Fax 0722 303401 > > > > > > > > > -----Messaggio originale----- > > > Da: methods-bounces@oat.bio.indiana.edu > > > [mailto:methods-bounces@oat.bio.indiana.edu] Per conto di > > > kjaanson@gmail.com > > > Inviato: domenica 10 febbraio 2008 21.57 > > > A: methods@magpie.bio.indiana.edu > > > Oggetto: CHEF gel genomic DNA restriction(No virus check: scan engine > > > not ready) > > > > > > Hi > > > > > > I have lately tried to restrict the genomic DNA of mammalian cells in > > > agarose plugs and then separate the fragments with CHEF pulse field. > > > The restriction seems to be working but the restricted DNA migrates > > > weird in gel. It is very dense in the Kb-Mb range (EtBr staining shows > > > that the DNA in that range has kind of spread over the lane borders). > > > After the dense part ends the DNA occupies only the central part of > > > the lane. Southern plot of the gel shows that the DNA runs higher > > > (more slowly) than it should be, also the bands are a bit distorted. > > > > > > I was wondering if the anomaly might be associated with the > > > concentration of DNA in agarose plug, to my calculations there should > > > be about 3ug of genomic DNA in one plug. If that is the case then > > > could I vary the pulse field parameters so that the high amount of > > > genomic DNA would migrate normally? > > > > > > > > > Kaur Jaanson > > > _______________________________________________ > > > Methods mailing list > > > Methods@net.bio.net > > > http://www.bio.net/biomail/listinfo/methods > > > > > > > > > > > > > > -- > > > > > > > > Kaur Jaanson > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God -- Kaur Jaanson From sudhee26 from gmail.com Mon Feb 11 23:43:57 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Tue Feb 12 12:30:26 2008 Subject: R: CHEF gel genomic DNA restriction(No virus check: scan engine not ready) In-Reply-To: <4f6282d40802110135m267c881p8a83dc5777e3f01e@mail.gmail.com> References: <2a40d57d-b1c2-4e97-b458-a13271d6d03e@i12g2000prf.googlegroups.com> <001001c86c8f$d05e6730$7d0ca8c0@iram.it> <4f6282d40802110135m267c881p8a83dc5777e3f01e@mail.gmail.com> Message-ID: Hey, i found this one on net. check if it can troubleshoot some of your problems. I think looking again into your protocol would do lot of help. http://humgen.wustl.edu/hdk_lab_manual/12/12_2.html Sudheendra. On Feb 11, 2008 3:05 PM, Kaur Jaanson wrote: > Hi > > Sorry, forgot to add the conditions. > > I use 1.2% agarose gel with 0.5x TAE buffer, running parameters are > 5.4 V/cm field strength, 2-90 sec switch time, 19 hr run time. > Biorad CHEF DR-II system to run the pulse field electrophoresis. > > Kaur Jaanson > > On Feb 11, 2008 11:23 AM, Prof. Piero Sestili > wrote: > > Dear Kaur, > > > > post the running conditions you're using then we can talk about the CHEF > > protocol modifications. > > > > Cheers > > > > Prof. Piero Sestili > > Istituto di Farmacologia e Farmacognosia e > > Centro di Ricerca sull'Attivit? Motoria > > Universit? degli Studi di Urbino "Carlo Bo" > > Via "I Maggetti" 26 > > 61029 URBINO (PU) > > Tel. 0722 303414; 0722 305524 > > Fax 0722 303401 > > > > > > -----Messaggio originale----- > > Da: methods-bounces@oat.bio.indiana.edu > > [mailto:methods-bounces@oat.bio.indiana.edu] Per conto di > > kjaanson@gmail.com > > Inviato: domenica 10 febbraio 2008 21.57 > > A: methods@magpie.bio.indiana.edu > > Oggetto: CHEF gel genomic DNA restriction(No virus check: scan engine > > not ready) > > > > Hi > > > > I have lately tried to restrict the genomic DNA of mammalian cells in > > agarose plugs and then separate the fragments with CHEF pulse field. > > The restriction seems to be working but the restricted DNA migrates > > weird in gel. It is very dense in the Kb-Mb range (EtBr staining shows > > that the DNA in that range has kind of spread over the lane borders). > > After the dense part ends the DNA occupies only the central part of > > the lane. Southern plot of the gel shows that the DNA runs higher > > (more slowly) than it should be, also the bands are a bit distorted. > > > > I was wondering if the anomaly might be associated with the > > concentration of DNA in agarose plug, to my calculations there should > > be about 3ug of genomic DNA in one plug. If that is the case then > > could I vary the pulse field parameters so that the high amount of > > genomic DNA would migrate normally? > > > > > > Kaur Jaanson > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > > > -- > Kaur Jaanson > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From hzhen from freeuk.com Tue Feb 12 15:42:38 2008 From: hzhen from freeuk.com (ChenHA) Date: Tue Feb 12 18:21:02 2008 Subject: Kd semantics In-Reply-To: <8SYrj.7610$iB4.460@newsfe07.lga> References: <8SYrj.7610$iB4.460@newsfe07.lga> Message-ID: <1202849253.8712.0@proxy01.news.clara.net> DK wrote: > In article , kaj.stenberg@helsinki.fi.invalid wrote: >> DK wrote: >>> In article , >> kaj.stenberg@helsinki.fi.invalid wrote: >>>> This is probably trivial, but it confuces me: >>>> >>>> If the Kd for a ligand is mM it binds poorly, if the Kd is nM it binds >>>> well. But in which case does one refer to the Kd as being "higher" or >>>> "lower"? >>> mM is "higher" than nM. However, sometimes people switch from >>> talking about Kd to talking about "affinity". Affinity is "higher" when >>> Kd ~ nM. Yes, people tend to say "X has a high affinity binding with a Kd of ...", the reason probably being that affinity is easily understood semantically, while the number from Kd is easily understood scientifically. No problem if you know what affinity is, but many student don't. >> Thank you, "affinity" is of course unambigious, I did just not come to >> think of it. >> >> Just another thing: when can you start talking about a "real" (as in >> specific) affinity? I am reading papers with a Kd in the ballpark of >> tens of millimolar and find it hard to repeat the work so that I truly >> believe in the results, since every random event seems to affect the >> curves fairly much. > > AFAIK, for proteins > mM affinities are usually derived indirectly > from some other data (such as Km) rather than measured. > > There can never be a strict threshold but for protein-protein > interaction the fuzzy boundary between real and not, specific > and not lies somewhere around tens of microM Kd. The bane of my life, trying to find out whether the binding of a protein or peptide is real or has any significance. BTW, I had done experiments where I showed that a binding in the low mM range is real and specific (by NMR - you can show specific binding by the effect on particular cluster of residues). Whether it has any significance is another matter entirely. > > > > From hzhen from freeuk.com Tue Feb 12 16:51:19 2008 From: hzhen from freeuk.com (ChenHA) Date: Tue Feb 12 18:21:15 2008 Subject: Kd semantics In-Reply-To: References: <8SYrj.7610$iB4.460@newsfe07.lga> <1202849253.8712.0@proxy01.news.clara.net> Message-ID: <1202853373.22671.0@demeter.uk.clara.net> DK wrote: > In article <1202849253.8712.0@proxy01.news.clara.net>, ChenHA wrote: >> DK wrote: >>> There can never be a strict threshold but for protein-protein >>> interaction the fuzzy boundary between real and not, specific >>> and not lies somewhere around tens of microM Kd. >> The bane of my life, trying to find out whether the binding of a protein >> or peptide is real or has any significance. BTW, I had done experiments >> where I showed that a binding in the low mM range is real and specific >> (by NMR - you can show specific binding by the effect on particular >> cluster of residues). Whether it has any significance is another matter >> entirely. > > Thanks for joining my club :-) > > My reasoning about tens of uM follows simply from the fact that few > proteins have intracellular concentration exceeding that. (Calmodulin, > which is pretty abundant, is estimated to be around 30 uM; actin > is not much higher, I think). So, unless the process is exlusively under > kinetic control (I think this is rather rare), it would have to work at > this concentrations - which places a high limit on the Kd. That particular protein I worked on (years ago) is a membrane surface protein which has a tendency to cluster (there's other bane of my life, trying to stop protein aggregation messing up true binding), so the local concentration at the interaction site may be very high. Doubtful it has any real significance, but you never know. > > (Then, of course, there is always a possibility of molecule C > significantly affecting interaction between A and B, so people > tend to always assume some C really does exist...) > > DK From josenet from tiscali.co.uk Tue Feb 12 19:59:54 2008 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Wed Feb 13 10:23:59 2008 Subject: site directed mutagensis References: Message-ID: <61etvgF1h1g9eU1@mid.individual.net> "doaa zineldeen" wrote in message news:mailman.587.1202621439.2451.methods@net.bio.net... > hello > I just want if anyone have a protocol for pcr mediated site directed > mutagensis without using stratagene kit > i have a template vector containing the insert and i want to mutate k81 > into > A i designed 2 primers flanking the mutation site and i want can i perform > 2 > pcr steps and a third mix with normal polymerase i am using phusion > can i proceed to get the new product containing the mutated sites > If someone can help i am really grateful > Doaa For a point mutation (or a couple) the "Quickchange" PCR system works well. I know you don't want to use Stratagene's kit... and neither do I. But you don't need any kit to use the strategy. Read their manual to get a few hints, and do it with your own reagents... I just sequenced 3 clones out of 8 I picked for a double point mutation (vector+insert size = 6kb)... all three were perfect. Just one step. For other things I use a "megaprimer" approach (google) which is another simple strategy. Jose From Vince.Mulholland from sasa.gsi.gov.uk Wed Feb 13 04:44:00 2008 From: Vince.Mulholland from sasa.gsi.gov.uk (Vince Mulholland) Date: Wed Feb 13 10:24:15 2008 Subject: Enterohaemorrhagic Escherichia coli Message-ID: >Dear Sir, > > I am Pooja, a student at IISc, India. I was reading about pathogenic E.coli and >just had a simple query. Sir, do you need any special facility to work with >pathogenic E.coli like 0155:H7 or can we work in the normal hood used for >culturing nonpathogenic E.coli. Are any specific precautions required while >working with pathogenic E.coli? > >Waiting eagerly for your reply. > > Regards, > Pooja Just a word of caution. As a student you should only undertake this kind of work under the direct supervision of someone experienced in handling pathogenic bacteria. That supervision should give you the theoretical and practical skills required to handle the bacteria in a contained fashion. If this is a new area of work for the laboratory (no matter if there are workers who have used pathogenic bacteria elsewhere) then the advice of a departmental or institutional biological safety officer should be obtained before commencing the work. The BSO should be able to give guidance on national rules, but will also be able to ensure you comply with the local rules of your institute. Remember when working with pathogens you have a duty of care to yourself and your family, your co-workers, other staff (such as cleaners, porters and engineers) and the wider community outside the institute. Regards, Vince Mulholland Correspondents should note that all communications to or from the Scottish Agricultural Science Agency may be automatically logged, monitored and/or recorded for lawful purposes. The original of this email was scanned for viruses by the Government Secure Intranet (GSi) virus scanning service. On leaving the GSi this email was certified virus-free. From kiraithem from gmail.com Wed Feb 13 04:45:42 2008 From: kiraithem from gmail.com (kiraithe michael) Date: Wed Feb 13 10:24:21 2008 Subject: experimeriment protocols Message-ID: <3a5c26090802130145p68eecc7xfce53eb64e36f913@mail.gmail.com> I am an upcoming research scientist requesting your invaluabe help in protocols to investigate the serum levels of cholestrol, SOD, GST, and any other that may help me to establish the oxidative stress levels in maternal and fetal serum samples. From johncumbers from gmail.com Wed Feb 13 22:06:53 2008 From: johncumbers from gmail.com (John Cumbers) Date: Thu Feb 14 14:04:42 2008 Subject: Why are qPCR reagents so expensive and is there a cheaper option? Message-ID: Hi, I'm trying to teach qPCR in a lab, ok the machine is really expensive (Ab7300) but what is it about the reagents that make them cost so much (e.g bio-rad, itaq SYBR supermix + ROX) it costs about $100 per 96 well plate. Is it because the reagents are patented still? Can I make my own? Apart from lower reaction volumes, is there anything else I can do to reduce the costs? Also are there more affordable qPCR machines on the market these days? The ab7300 is quite old now I think. Cheers, John -- John Cumbers, Graduate Student Molecular Biology, Cell Biology, and Biochemistry Biology and Medicine Brown University, Box G-W Providence, Rhode Island, 02912, USA Tel USA: +1 401 523 8190, Fax: +1 401 863-2166 UK to USA: 0207 617 7824 From stxsz1 from nottingham.ac.uk Thu Feb 14 08:14:48 2008 From: stxsz1 from nottingham.ac.uk (Zhong Silin) Date: Thu Feb 14 14:05:22 2008 Subject: what is the most quantitative method for SNP detection Message-ID: Hi everyone If I have a mixture of 2 DNA, which has just one SNP, say 40% GGG and 60% GTG. What SNP detection method is the best to quantify it? Is the Beckman SNP detection method the best? I heard that their kit is similar to a dye terminator sequence reaction. The reaction doesn't contain normal dNTP. So only 1 bp can be extended and therefore the primer will link to one dye-ddNTP. Maybe this will generate less background than a normal sequencing? Is there a better way? Thanks in advance silin This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation. From JANSSEDE from student.gvsu.edu Thu Feb 14 12:45:17 2008 From: JANSSEDE from student.gvsu.edu (Derek H. Janssens) Date: Thu Feb 14 14:11:25 2008 Subject: SyBR green home mix Message-ID: <1203011117.95ccd29cJANSSEDE@student.gvsu.edu> Hello, I was wondering if you were ever able to create an effective SyBR green home mix, and if so I was hoping that you could tell me what you use. Also I have been having trouble finding a company that sells SyBR green dye independent of a kit. Do you know of one? Thank You Derek Janssens From R.Jayakumar from roswellpark.org Thu Feb 14 16:49:44 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Thu Feb 14 19:35:32 2008 Subject: Why are qPCR reagents so expensive and is there a cheaper option? In-Reply-To: References: Message-ID: <97101976F8A044468CA74FE11883B90E173E79C9@VISTA.roswellpark.org> You could try the cheaper QPCR one color systems from Bio-RAD. They are good enough for small labs. The ABI systems (AB7300 and above) are too expensive for a small college lab. The Sybrgreen mixes should be available cheaper from other companies (search on Biocompare.com). Or you can make your own kit (you should get the recipes from the web)from sybr green and rox dyes available from Sigma or some other similar companies. Best of luck Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of John Cumbers Sent: Wednesday, February 13, 2008 10:07 PM To: Methods@magpie.bio.indiana.edu Subject: Why are qPCR reagents so expensive and is there a cheaper option? Hi, I'm trying to teach qPCR in a lab, ok the machine is really expensive (Ab7300) but what is it about the reagents that make them cost so much (e.g bio-rad, itaq SYBR supermix + ROX) it costs about $100 per 96 well plate. Is it because the reagents are patented still? Can I make my own? Apart from lower reaction volumes, is there anything else I can do to reduce the costs? Also are there more affordable qPCR machines on the market these days? The ab7300 is quite old now I think. Cheers, John -- John Cumbers, Graduate Student Molecular Biology, Cell Biology, and Biochemistry Biology and Medicine Brown University, Box G-W Providence, Rhode Island, 02912, USA Tel USA: +1 401 523 8190, Fax: +1 401 863-2166 UK to USA: 0207 617 7824 _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. From usiva from ufl.edu Thu Feb 14 15:45:45 2008 From: usiva from ufl.edu (uthandi sivakumar) Date: Thu Feb 14 20:36:37 2008 Subject: Request protocol for Genomic DNA extraction from Gram +ve Message-ID: <000001c86f4a$930c77b0$b410f90a@ad.ufl.edu> I am in need a protocol for genomic dna isolation from actinomycets SIva From beakerhead from live.com Thu Feb 14 16:52:12 2008 From: beakerhead from live.com (beakerhead testtube) Date: Thu Feb 14 20:36:45 2008 Subject: Thin Layer Chromatography Help Message-ID: I'm looking for an reservior and applicator for thin layer chromatography. Our lab has SOP's developed using the reservoir in the attached picture, but we don't have the manufacturer or part number. We're also looking for the applicators that fit in the smaller slots around the central slot. If anyone can help, it would be much appreciated. _________________________________________________________________ Shed those extra pounds with MSN and The Biggest Loser! http://biggestloser.msn.com/ From blackhole from abuse.plus.com Fri Feb 15 04:52:20 2008 From: blackhole from abuse.plus.com (Duncan Clark) Date: Fri Feb 15 12:02:37 2008 Subject: Why are qPCR reagents so expensive and is there a cheaper option? References: Message-ID: Historians believe that in newspost on Wed, 13 Feb 2008, John Cumbers penned the following literary masterpiece: >what is it about the reagents that make them cost so much (e.g >bio-rad, itaq SYBR supermix + ROX) it costs about $100 per 96 well plate. >Is it because the reagents are patented still? Can I make my own? Patent on antibody so royalty payable. If chemically modified enzyme, patent on that so again royalty payable Patent on SYBR so royalty payable Patent(s) on qPCR so royalty payable, both on hardware hence the instrument price, and reagents. Basically royalty stacking is a big part of the problem, a few percent for each bit adds up to quite a total. I doubt it is far short of 20-30%. It also doesn't help that there are very very few alternatives to SYBR. Naturally you can make up your own mixes and over the years I've seen quite a few recipes published. Google should find them. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From blackhole from abuse.plus.com Fri Feb 15 04:56:44 2008 From: blackhole from abuse.plus.com (Duncan Clark) Date: Fri Feb 15 12:02:42 2008 Subject: SyBR green home mix References: Message-ID: Historians believe that in newspost on Thu, 14 Feb 2008, Derek H. Janssens penned the following literary masterpiece: > SyBR green dye independent of a kit. Do you know of one? SYBR Green I is patented and owned by Molecular Probes who are part of the Invitrogen group. It's available off the shelf direct from Invitrogen. Cat. No. S7563 at around GBP167 for 500ul of a 10000x stock. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From scrampto from uci.edu Fri Feb 15 13:49:12 2008 From: scrampto from uci.edu (Steve Crampton) Date: Fri Feb 15 17:49:43 2008 Subject: SyBR green home mix (Duncan Clark) Message-ID: <3F4C4375-8A5A-4F2D-9B3A-FE50EE407614@uci.edu> We use our own QPCR MM that includes the SyBR green from Molecular probes. This home made mix is much cheaper than premade mixes from most companies. the reference for the mix is below the recipe. Of course, you still have to get the Hot start taq (we use Qiagens). Steve Quantitative PCR Master Mix (2X) makes 10mL store in aliquots in the -20 Stock reagent Volume (10mL) Final 1M Tris pH 8.3 (UV irradiated) 1mL 100mM 25mM MgCL2 (Qiagen) 2.4mL 6mM 10mg/mL BSA Omnipure Fraction V 1mL 1mg/mL 10mM dNTP Mix 400?L 400?M 100X SyBR Green 66.67 ?L 0.66X dH2O (irradiated DEPC) 5.032 mL **for use: Add 10?L Qiagen HotStarTaq per 1mL Master Mix Rxn 10?L MM 5?L dH2O 2?L upper primer 2?L lower primer 1?L template 20?L Reference for MM Phosphoinositide 3-kinase and Bruton's tyrosine kinase regulate overlapping sets of genes in B lymphocytes. Fruman DA, Ferl GZ, An SS, Donahue AC, Satterthwaite AB, Witte ON Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):359-64 From scrampto from uci.edu Fri Feb 15 13:56:43 2008 From: scrampto from uci.edu (Steve Crampton) Date: Fri Feb 15 17:49:50 2008 Subject: Qiagen high speed maxi Message-ID: <1178908C-AF14-4E4C-B114-EAE19F2960BB@uci.edu> We have been using the Qiagen high speed maxi prep kit to isolate plasmid DNA for a while and have had some problems. We have standardized our growth time and pretty much always prep high copy plasmids such as PcDNA. The problem is that sometimes we get 1 mg of DNA and sometimes we get no DNA at all! My wife (in a separate lab) has also had this problem and they did a side by side comparison (2 different columns, same cultures, etc) and one had 0.8mg and the other no DNA. Has anyone else had a similar yield problem with this kit? Steve Crampton From sudhee26 from gmail.com Sat Feb 16 00:47:19 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Sat Feb 16 13:23:28 2008 Subject: Qiagen high speed maxi In-Reply-To: References: Message-ID: hi, check for the common lot number??? the dna yeild from qiagen columns are not going to be same from every column..however they will surely not differ drastically. i have used QIA100, 500 but not 1000. best, sudheendra. On Sat, Feb 16, 2008 at 8:07 AM, DK wrote: > In article , Steve > Crampton wrote: > >We have been using the Qiagen high speed maxi prep kit to isolate > >plasmid DNA for a while and have had some problems. We have > >standardized our growth time and pretty much always prep high copy > >plasmids such as PcDNA. The problem is that sometimes we get 1 mg of > >DNA and sometimes we get no DNA at all! My wife (in a separate lab) > >has also had this problem and they did a side by side comparison (2 > >different columns, same cultures, etc) and one had 0.8mg and the other > >no DNA. Has anyone else had a similar yield problem with this kit? > > That's pretty odd. Of all things, the columns would be the last > thing I'd blame. You can be 99.999% sure they are packed with > the same ion exchanger! > > Is it possible some of them are not wetted properly? If I suspected > this being the case, I'd wash the column with ethanol and then water > before proceeding with the protocol. > > Personally, I've never experienced any yield problems with > Qiagen's Maxi preps. > > DK > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From Nikola.Wenta from nottingham.ac.uk Sun Feb 17 12:48:17 2008 From: Nikola.Wenta from nottingham.ac.uk (Wenta Nikola) Date: Sun Feb 17 13:41:05 2008 Subject: Qiagen high speed maxi (Sudheendra Rao N R) Message-ID: Hello! Is the column for the maxi prep a silica membrane like for other DNA purification kits? If so, then different yields could be explained easily because the silica gel is a natural product of varying quality. But anyway, it should not be possible that special lots lead to zero-yield but lower yields. Cheers, Niko This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation. From Nikola.Wenta from nottingham.ac.uk Sun Feb 17 12:52:36 2008 From: Nikola.Wenta from nottingham.ac.uk (Wenta Nikola) Date: Sun Feb 17 13:41:15 2008 Subject: site directed mutagensis (Jose de las Heras) Message-ID: Hello! In our lab, we use Stratagene's Pfu Turbo with up to 14 mins elongation time (annealing temoerature is important!). Then digest with DpnI and transformation into XL2-Blue. Works fine! Cheers, Niko This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation. From engelbert_buxbaum from hotmail.com Mon Feb 18 10:55:14 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Feb 18 14:29:09 2008 Subject: MTT References: <031501c8642a$b35b7aa0$2e7a01a3@dpag.ox.ac.uk> Message-ID: Am 01.02.2008, 17:09 Uhr, schrieb Tom Anderson : > I think if i had access > to a Coulter counter, i'd be using that instead, and getting direct > measurements of cell number and volume. The problem is that a Coulter counter does not distinguish between dead and alive cells. Also, it tends to be more work. And plate readers are much more wide spread than Coulter counters. >> so if I treat the cells with a compound how am I supposed to know if >> it's doing anything to their rate of proliferation? > > You need a standard every time you do the experiment. I'm not sure if you > need a whole standard curve, or just a single control well, where you > haven't added a test compound. The latter should be fine if the assay's > linear. Maybe three control wells, to be sure. The usual way is to do a concentration dependency: Suspend your cells in 20 ml medium and pipette 0.2 ml aliquots into each well (taking care that the cells can not settle). Allow cells to attach o/n, remove the old medium and replace with 150 ul fresh. In the wells numbered H, add 150 ul stock solution of the chemicals you want to test, mix gently. Take 150 ul to well G, mix gently and take 150 ul to well F. Continue until you have reached well B, where you discard the excess 150 ul. With this procedure you get a series of different concentrations of your chemicals, doubling from well to well, except in well A where the concentration is 0. Let the cells grow until they approach confluency in the well A, then perform your assay. Plot the absorbance you get against the the concentration of your chemical, you should get a S-shaped curve starting at 100% growth in the first wells. The relevant parameter is the concentration at which you have only 50% growth, this is called IC50 and obtained by non-linear curve fitting. These IC50 values you can use to compare the toxicity of different compounds on the same cell type, the same compound on different cell types, or the same compounds on the same cells under different conditions. It is best to do the assays in triplicates, so you can test 4 different chemicals in each plate. From engelbert_buxbaum from hotmail.com Mon Feb 18 11:12:25 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Feb 18 14:29:16 2008 Subject: Can you heat RPMI 1640? References: Message-ID: Am 07.02.2008, 08:39 Uhr, schrieb Kamalian, Laleh : > I have found the concentration of the components and every thing else. > The only problem is how to dissolve methylcellulose in the media. I > found out that I have to add warm (60 ' C) liquid to the powder and let > it cool down while stirring. But can I heat RPMI 1640 media to 60"? I > couldn't find the answer on the net. I would produce a 10 or 20 x stock solution of Methylcellulose in water and autoclave to get it sterile. Then use 10 x RPMI (commercially available or prepared from powder) to make the final medium. From engelbert_buxbaum from hotmail.com Mon Feb 18 15:07:08 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Feb 18 16:17:02 2008 Subject: Kd semantics References: Message-ID: Am 11.02.2008, 03:50 Uhr, schrieb : > Just another thing: when can you start talking about a "real" (as in > specific) affinity? I am reading papers with a Kd in the ballpark of > tens of millimolar and find it hard to repeat the work so that I truly > believe in the results, since every random event seems to affect the > curves fairly much. Remember that in order to get accurate results for binding studies both [E] and [S] should be in the same order of magnitude as Kd. Thus for Kd = 1 mM -> [E] should be at least 0.1 mM (better several times more), and as you can calculate yourself that is quite a lot for a substance of say, M = 50 kDa. From engelbert_buxbaum from hotmail.com Mon Feb 18 15:26:41 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Feb 18 16:17:16 2008 Subject: loooking for protein phosphorylation method References: Message-ID: Am 12.02.2008, 04:28 Uhr, schrieb prabhat khadka : > hi > If anyone could help me with protein phosphorylation protocal , taht > will be highly appericated. > hope to see soon If you provided a little more info on what you want to do it would be easier to help. So looking into my crystal ball I'd guess you want to metabolically label proteins with 32P (or 33P, which I personally prefer unless double- or triple labelling is required). In that case you incubate the cells in medium containing the isotope in the form of phosphate, with as much label as local regulation allows you to use in a single reaction. There should be no other source of P in the medium, but you need glucose to get that phosphate into ATP. After incubation (time and conditions depend on the experiment) spin, wash and homogenise the cells (for the small volume involved a ball-bearing homogeniser is nice, but a Potter will also do). The next step involves isolation of the compartment you want to investigate, usually by fractionated centrifugation. Any compartment other than cytosol would be "opend" in then. Soluble proteins are separated from small molecules (which contain most of the radioactivity) by gel filtration on a small, disposable column. Membrane proteins are separated by centrifugation instead. Next you separate the proteins, e.g. by 2D-electrophoresis followed by autoradiography. If you can do that with a phosphor-imager you already have quantitative results, otherwise you have to cut out the spots of interest, digest the gel and count the radioactivity in a beta-counter. From engelbert_buxbaum from hotmail.com Mon Feb 18 16:18:23 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Feb 18 17:38:18 2008 Subject: experimeriment protocols References: Message-ID: Am 13.02.2008, 05:45 Uhr, schrieb kiraithe michael : > I am an upcoming research scientist requesting your invaluabe help in > protocols to investigate the serum levels of cholestrol, SOD, GST, and > any > other that may help me to establish the oxidative stress levels in > maternal > and fetal serum samples. Those are standard assays described in any textbook of clinical chemistry. A trip to your library should provide the required info. From engelbert_buxbaum from hotmail.com Mon Feb 18 16:24:33 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Feb 18 17:38:30 2008 Subject: Thin Layer Chromatography Help References: Message-ID: Am 14.02.2008, 17:52 Uhr, schrieb beakerhead testtube : > > I'm looking for an reservior and applicator for thin layer > chromatography. Our lab has SOP's developed using the reservoir in the > attached picture, but we don't have the manufacturer or part number. Pictures don't work here, this group is text only. But you should be able to get chromatography equipment from the usual lab suppliers (Fisher, BDH, Sigma...) From wxfhome from gmail.com Tue Feb 19 04:41:23 2008 From: wxfhome from gmail.com (wxfhome@gmail.com) Date: Tue Feb 19 13:18:32 2008 Subject: Inducer of apoptosis in animal Message-ID: <47f74ecd-2335-4171-8ee6-963c78c4789d@e10g2000prf.googlegroups.com> Hello! I want to test a method which can detect apoptosis in mice, and I do not know how to induce apoptosis in mice. Could anyone give some suggestions about the inducer and protocol? I see H2O2 can induce apoptosis in cell culture. Thank you all. LW From legatek from hotmail.com Tue Feb 19 13:19:25 2008 From: legatek from hotmail.com (Kyle Legate) Date: Tue Feb 19 16:47:11 2008 Subject: Inducer of apoptosis in animal In-Reply-To: <47f74ecd-2335-4171-8ee6-963c78c4789d@e10g2000prf.googlegroups.com> References: <47f74ecd-2335-4171-8ee6-963c78c4789d@e10g2000prf.googlegroups.com> Message-ID: <620kt8F20kj81U1@mid.individual.net> wxfhome@gmail.com wrote: > Hello! > > I want to test a method which can detect apoptosis in mice, and I do > not know how to induce apoptosis in mice. Could anyone give some > suggestions about the inducer and protocol? I see H2O2 can induce > apoptosis in cell culture. > I'm not sure that inducing apoptosis in a whole mouse would pass ethics boards. I know they wouldn't see it as necessary. Select a tissue which is known to have a significant amount of natural apoptosis, like the skin during hair follicle catagen (around 8 weeks after birth) or the mammary gland sometime after weaning, during involution, and test your method on those samples. From pow.joshi from gmail.com Tue Feb 19 13:47:59 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Tue Feb 19 16:47:20 2008 Subject: Inducer of apoptosis in animal In-Reply-To: <47f74ecd-2335-4171-8ee6-963c78c4789d@e10g2000prf.googlegroups.com> References: <47f74ecd-2335-4171-8ee6-963c78c4789d@e10g2000prf.googlegroups.com> Message-ID: <710764ea0802191047j556899c5h93fe88a5c75804bf@mail.gmail.com> On 19/02/2008, wxfhome@gmail.com wrote: > > Hello! > > I want to test a method which can detect apoptosis in mice, and I do > not know how to induce apoptosis in mice. Could anyone give some > suggestions about the inducer and protocol? I see H2O2 can induce > apoptosis in cell culture. hello, you mean you want to induce apoptosis in vivo? Does it have a specific region of an organ system? Because, an apoptosed mouse, I'd imagine is a very dead mouse. Pow Thank you all. > > LW > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From novalidaddress from nurfuerspam.de Tue Feb 19 17:07:30 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Feb 19 20:32:37 2008 Subject: Inducer of apoptosis in animal References: <47f74ecd-2335-4171-8ee6-963c78c4789d@e10g2000prf.googlegroups.com> Message-ID: <3a5cd921-ee77-48f8-ae19-5014ccb3ac7f@p73g2000hsd.googlegroups.com> > Because, an apoptosed mouse, I'd > imagine is a very dead mouse. > > Pow Yes, very dead indeed. In German, this is called "mausetot" (as dead as a mouse...) What about taking out the organs of interest, perfusing them like in the Langendorf Heart Model and include apoptosis inducers in the buffer? As lethal to mice as whole animal apoptosis, but a bit more human... Best, Wo From wxfhome from gmail.com Wed Feb 20 00:01:17 2008 From: wxfhome from gmail.com (WANG XF) Date: Wed Feb 20 12:54:02 2008 Subject: Inducer of apoptosis in animal In-Reply-To: <710764ea0802191047j556899c5h93fe88a5c75804bf@mail.gmail.com> References: <47f74ecd-2335-4171-8ee6-963c78c4789d@e10g2000prf.googlegroups.com> <710764ea0802191047j556899c5h93fe88a5c75804bf@mail.gmail.com> Message-ID: <404daa2f0802192101kf01644y912096427d779a23@mail.gmail.com> Hi, Thanks a lot for your help! Yes, I want to induce and observe apoptosis in vivo, and the apoptosis should take place at some tissues or regions (subcutaneous or liver, et al.), but not the whole mouse. LW On 2/20/08, Pow Joshi wrote: > > > > On 19/02/2008, wxfhome@gmail.com wrote: > > > > Hello! > > > > I want to test a method which can detect apoptosis in mice, and I do > > not know how to induce apoptosis in mice. Could anyone give some > > suggestions about the inducer and protocol? I see H2O2 can induce > > apoptosis in cell culture. > > > > hello, you mean you want to induce apoptosis in vivo? Does it have a > specific region of an organ system? Because, an apoptosed mouse, I'd > imagine is a very dead mouse. > > Pow > > > Thank you all. > > > > LW > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > From wxfhome from gmail.com Wed Feb 20 00:29:03 2008 From: wxfhome from gmail.com (WANG XF) Date: Wed Feb 20 12:54:17 2008 Subject: Lipo2000 leads to cell apoptosis? Message-ID: <404daa2f0802192129u21873d8ev2b9a6e9b87bb2159@mail.gmail.com> Usually transfection was completed with lipofectamine 2000 in our lab. I am detecting apoptosis induced with H2O2, and after transfection of the apoptosis sensor construct into cells, apoptosis could be found in the absense of H2O2. lipofectamine 2000 or other transfection reagents based on liposome will lead to cell apoptosis? Thanks. LW From huang_wen from ustc.edu Wed Feb 20 12:45:14 2008 From: huang_wen from ustc.edu (Wen Huang) Date: Wed Feb 20 12:55:04 2008 Subject: bisulfite dna modification kit Message-ID: <69496F7E-B890-4C55-9D7B-119693456A12@ustc.edu> Hi there, I am just asking for suggestions on bisulfite dna modification kit, anybody knows some efficient, easy-to-use, not very expensive kit? I am thinking about the following, anybody can provide reviews? Millipore CpGenome DNA modification kit Sigma Imprint DNA modification kit Epigentek Methylamp DNA modification kit thanks, wen From yvonne.couch from dpag.ox.ac.uk Wed Feb 20 12:08:10 2008 From: yvonne.couch from dpag.ox.ac.uk (Yvonne Couch) Date: Wed Feb 20 12:55:12 2008 Subject: pRRL Message-ID: <004a01c873e3$2d09b340$2e7a01a3@dpag.ox.ac.uk> Hi all, I have the lentiviral parent vector pRRL-GFP but have no map for it. I have the sequence and I need to know whether I can perform LR recombination but am not really sure how to find the attR and attL sites in the sequence. Can anybody help? Cheers Yvonne From ebs15242 from creighton.edu Wed Feb 20 13:56:30 2008 From: ebs15242 from creighton.edu (Ed Siefker) Date: Wed Feb 20 14:25:42 2008 Subject: low plasmid yields in dh5alpha Message-ID: <47BC77DE.1000204@creighton.edu> Hi. We've been going through our old plasmids and making fresh stocks. We've found a few that we're not able to miniprep and get reasonable concentrations (<50ng/ul). We're using Qiagen miniprep kits and invitrogen dh5alpha and have had much success purifying One thought was that the plasmids might be producing a product that is toxic to dh5alpha, and maybe they'd do better in another strain. Has anyone had a similar problem that was resolved by growing the plasmid in another strain? I'm thinking XL1-blue might help because it retains the lac repressor, and that might help stop any background expression from the lac promoter. Any other ideas would be appreciated. Thanks -Ed From novalidaddress from nurfuerspam.de Wed Feb 20 18:1