From rahimpour_a from yahoo.com Mon Nov 2 12:32:15 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Mon Nov 2 14:51:25 2009 Subject: tm calculation for tagged primers Message-ID: <445419.87768.qm@web52701.mail.re2.yahoo.com> Hi I am new to primer design and I have a simple question: when I add extra sequences like restriction sites and epitope tags to my primer, should I calculate primer tm with these sequences or not? actually when I calculate Tm with these sequences the tm is about 80 and if I ignore them it seems that the calculation isnt complete. is there any rule for that? regards From cjmcdermottroe from googlemail.com Mon Nov 2 15:24:53 2009 From: cjmcdermottroe from googlemail.com (cjmcdermottroe@gmail.com) Date: Mon Nov 2 16:09:48 2009 Subject: tm calculation for tagged primers Message-ID: <21039324.13196.1257193494712.JavaMail.seven@ap1.p2.uk.7sys.net> based on the definition of tm, it follows that since binding will not (or at least should not) occur between the template and your added restriction site, you should not therefore factor it in when calculating tms, gc etc. Fyi, adding flag/myc tags etc on a primer will give you crazy tms. Again, no binding so dont worry about them. Gl -- I sent this from my 3 mobile -- -original message- Subject: tm calculation for tagged primers From: "Azam Rahimpour" Date: 02/11/2009 7:53 pm Hi I am new to primer design and I have a simple question: when I add extra sequences like restriction sites and epitope tags to my primer, should I calculate primer tm with these sequences or not? actually when I calculate Tm with these sequences the tm is about 80 and if I ignore them it seems that the calculation isnt complete. is there any rule for that? regards _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From taliaferrod from mail.nih.gov Mon Nov 2 16:25:42 2009 From: taliaferrod from mail.nih.gov (Taliaferro, Dwayne (NIH/NIMH) [F]) Date: Mon Nov 2 19:06:29 2009 Subject: tm calculation for tagged primers In-Reply-To: <445419.87768.qm@web52701.mail.re2.yahoo.com> Message-ID: While there is some value in calculating TM and such, sometimes you just have to take a chance and order the primer and use it at 55-60C annealing temp. Primers are cheap! As for your particular question, I would calculate the TM for the part of the primer that will bind to the template and go from there. Also in my experience high TMs never hurt my PCR reactions, low TMs have though. Good luck On 11/2/09 12:32 PM, "Azam Rahimpour" wrote: Hi I am new to primer design and I have a simple question: when I add extra sequences like restriction sites and epitope tags to my primer, should I calculate primer tm with these sequences or not? actually when I calculate Tm with these sequences the tm is about 80 and if I ignore them it seems that the calculation isnt complete. is there any rule for that? regards _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From pjie2 from cam.ac.uk Tue Nov 3 07:31:05 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Tue Nov 3 10:34:15 2009 Subject: tm calculation for tagged primers In-Reply-To: References: Message-ID: On Mon, 2 Nov 2009, cjmcdermottroe@gmail.com wrote: > > based on the definition of tm, it follows that since binding will not > (or at least should not) occur between the template and your added > restriction site, you should not therefore factor it in when calculating > tms, gc etc. Fyi, adding flag/myc tags etc on a primer will give you > crazy tms. Again, no binding so dont worry about them. Gl To be more precise - during the first few cycles of PCR, you will be priming directly off your DNA template, and so the added parts will not match. For these cycles, you need to calculate the Tm based only on the matching parts of the primer (without your added tags). In later cycles, you are priming off the molecules synthesised during the earlier cycles, and the primer will thus match all the way along, including the tags. This means that if you want, you can do something similar to a touchdown PCR: use a lower Tm for the first few cycles, and then raise it. This may help if you are getting a lot of background / non-specific bands. However, I've never found this to be necessary, so I'd advise you to just calculate the lower Tm (i.e. without tags) and use that. Peter From allan.jones from gmx.de Tue Nov 3 11:58:41 2009 From: allan.jones from gmx.de (Allan Jones) Date: Tue Nov 3 14:18:31 2009 Subject: (no subject) Message-ID: <20091103165841.229520@gmx.net> Hi! Does anyone here know how stable Adenovirus (not replcation competent) is in mice? I was weighing out pulverized liver tissue of such mice and when cleaning my spatula afterwards with a wet cloth, splashed some of the liquid into my face (incl. lips). My labmates said that there will be very little to no intact virus in the animals after seven days,when the experiment ende (as it is not replication competent and should have either infected cells or been cleared by the mouse's immune system after this time). Does anyone here know if this isthecase, as i am a little worried about having an AV expressing shRNA in my cells. The doc in the building noted the accident, saying that replication incompetent viruses will not so any harm, especially Adenovirus, as it does not integrate, but i am still a little worried and unsure of how to handle this. Best Wishes! -- DSL-Preisknaller: DSL Komplettpakete schon f?r 16,99 Euro mtl.!* http://portal.gmx.net/de/go/dsl02 From stewjw from gmail.com Thu Nov 5 09:58:27 2009 From: stewjw from gmail.com (StewJW) Date: Thu Nov 5 12:19:05 2009 Subject: (no subject) References: Message-ID: <216d36a6-03a8-476d-8c9c-b1d8cb8ab6b1@n35g2000yqm.googlegroups.com> Shouldn't you have been doing this work in something like a class 2 cabinet? Anyhow to quote from a recent paper: Endogenous retroviruses (ERVs) represent the proviral phase of exogenous retroviruses that have integrated into the germ line of their host . They typically consist of an internal region with three genes (gag, pol, and env) plus two flanking, noncoding LTRs, which are identical at the time of integration. Human ERVs (HERVs) comprise ?5? 8% of the human genome. As long as it doesn't integrate into anywhere critical it'll just be one more on the list. Anyhow if its not replication competent surely that answers your question. I should add I know very little about this area but I did find your dilemma quite amusing. From RayHL from gmx.de Thu Nov 5 12:25:52 2009 From: RayHL from gmx.de (Thomas Schillinger) Date: Thu Nov 5 14:12:37 2009 Subject: =?iso-8859-1?q?Mold_problem_in_4=B0C_room?= Message-ID: <20091105172552.68130@gmx.net> Hello everyone, we have a problem with mold and its spores in our 4?C room. It was used as a storage room for several kinds of mold (in unwrapped/unsealed petri dishes) in the last few years while the former department was studying it. Now, this research has been discontinued and we are trying to use it as a "standard" cold room for storage of molecular biology reagents, protein purification purposes and so on. However, you can still smell the mold, and LB agar plates show signs of mold shortly after they have been put there. Does anybody know how to decontaminate it thoroughly? Or at least an idea how to do it? Thank you very much in advance! Greetings, Ray -- Jetzt kostenlos herunterladen: Internet Explorer 8 und Mozilla Firefox 3.5 - sicherer, schneller und einfacher! http://portal.gmx.net/de/go/chbrowser From hroychow from nmsu.edu Thu Nov 5 14:54:12 2009 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Thu Nov 5 17:53:47 2009 Subject: Mold problem in =?iso-8859-1?q?4=B0C_room?= In-Reply-To: <20091105172552.68130@gmx.net> References: <20091105172552.68130@gmx.net> Message-ID: <1948.128.123.174.0.1257450852.squirrel@webmail.nmsu.edu> We have used 8-10% bleach successfully on non-porous surfaces. Stainless steel benches and tables only should be allowed in a cold rooms. We have had to discard any wooden drawers or boxes (and of course those infernal cardboard boxes) from the cold rooms. We wiped the surfaces down and left the bleach solution over the weekend. > Hello everyone, > > we have a problem with mold and its spores in our 4?C room. It was used as > a storage room for several kinds of mold (in unwrapped/unsealed petri > dishes) in the last few years while the former department was studying it. > Now, this research has been discontinued and we are trying to use it as a > "standard" cold room for storage of molecular biology reagents, protein > purification purposes and so on. However, you can still smell the mold, > and LB agar plates show signs of mold shortly after they have been put > there. > > Does anybody know how to decontaminate it thoroughly? Or at least an idea > how to do it? > > Thank you very much in advance! > > > Greetings, > Ray > -- > Jetzt kostenlos herunterladen: Internet Explorer 8 und Mozilla Firefox 3.5 > - > sicherer, schneller und einfacher! http://portal.gmx.net/de/go/chbrowser > > _______________________________________________ > 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 swans061 from umn.edu Thu Nov 5 15:18:20 2009 From: swans061 from umn.edu (Debra Swanson) Date: Thu Nov 5 17:53:54 2009 Subject: =?iso-8859-1?q?Re=3A_Mold_problem_in_4=B0C_room?= In-Reply-To: <20091105172552.68130@gmx.net> References: <20091105172552.68130@gmx.net> Message-ID: <065063a557b1188e7ab44c146d1a4972@umn.edu> Greetings, We had this problem a couple years back in our walk-in cold rooms and the recommendation was to thoroughly clean the room, including walls and floor, with Dreft detergent. Also no paper/cardboard storage boxes are kept at 4? - plastic only. Good luck with the clean-up! Deb On Nov 5, 2009, at 11:25 AM, Thomas Schillinger wrote: Hello everyone, we have a problem with mold and its spores in our 4?C room. It was used as a storage room for several kinds of mold (in unwrapped/unsealed petri dishes) in the last few years while the former department was studying it. Now, this research has been discontinued and we are trying to use it as a "standard" cold room for storage of molecular biology reagents, protein purification purposes and so on. However, you can still smell the mold, and LB agar plates show signs of mold shortly after they have been put there. Does anybody know how to decontaminate it thoroughly? Or at least an idea how to do it? Thank you very much in advance! Greetings, Ray -- Jetzt kostenlos herunterladen: Internet Explorer 8 und Mozilla Firefox 3.5 - sicherer, schneller und einfacher! http://portal.gmx.net/de/go/chbrowser _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From pdeitik from bcm.tmc.edu Thu Nov 5 18:55:17 2009 From: pdeitik from bcm.tmc.edu (Deitiker, Philip R) Date: Thu Nov 5 19:26:52 2009 Subject: =?iso-8859-1?q?RE=3A_Mold_problem_in_4=B0C_room?= In-Reply-To: <1948.128.123.174.0.1257450852.squirrel@webmail.nmsu.edu> References: <20091105172552.68130@gmx.net> <1948.128.123.174.0.1257450852.squirrel@webmail.nmsu.edu> Message-ID: Mold grows in cold rooms because moisture collects on surfaces before being picked up by the evaporator coil on the refrigeration system. Many newer cold-rooms have a drying system on them to prevent the build-up of mold. They are expensive. In addition people who store live culture in the walk-in coolers have the most problem, because many volatile compounds can be effectively utilized by mold, and particular on wooden and paper surfaces. It is best to keep living culture wrapped or sealed tightly and to keep organic volatiles such as acetic acid, ammonia, amines, sulfides, etc sealed tightly. One can deal with mold as described below. If the wooden surfaces in the room are wood they have been varnished, the mold and moisture eventually destroys this and a surface such as Stainless steel or polyurethane treated wood may be preferable. Corion or stainless steel works well for table tops. One very common cause of mold is an improperly working fan-coil unit (the aspect of the machine that transfers air across the evaporator coil). This unit is supposed to pick up moisture and deliver that moisture to its condensate pan, were the condensate is rapidly drained. The condensate pan must be level on the outside edges and drain to a center position and quickly out of the cold-room. It is a frequent occurrence that the fan-coil unit is not doing this properly, the condensate tray builds up water and eventually waves form of the surface. The tops of these waves are picked up by lateral airflow and blow out the front of the unit as aspirate, basically causing all the surfaces in the walk-cooler to become permanently wet. Paper is a most effective collector of this moisture. -Causes of this can be: --Microbial clogging of the drain line. --A unit that is out of balance and vibrating excessively (such as a bad fan-motor bearing) --With volatile acids - the corrosion of the evaporator coil or the drain pan itself. --Other clogging agents. --Improperly installed or improperly working cooling fans (erratic icing over as another symptom) --A drain pan that is not properly leveled to expel excess water or blockage of the drain line for any other reason. Other causes: --Door left open excessively --Door seals are no longer separating outside (moist) air from inside (cold) air. A properly working cooler should not grow mold quickly, I cleaned out a cooler back in 1996 that had papered materials from 1984 stored on its shelf, most items did not have great amounts of mold on them. From naveenis4u from gmail.com Fri Nov 6 06:01:07 2009 From: naveenis4u from gmail.com (Naveen V) Date: Fri Nov 6 09:07:01 2009 Subject: =?iso-8859-1?q?Re=3A_Mold_problem_in_4=B0C_room?= In-Reply-To: <20091105172552.68130@gmx.net> References: <20091105172552.68130@gmx.net> Message-ID: <9cd5be270911060301m55e8733cp2dcf3db505ff774a@mail.gmail.com> Dear Thomas, The best, ideal and perfect way to abolish this problem is to fumigate your 4 degree/cold room. There are different ways to fumigate the best way is KMnO4 (Potassium permanganate) + Formaldehyde (the amount of addition will depend on size of your room) just i am illustrating the protocol 1. Vacate your room completely (the penetration and disinfection power is s= o high it can kill even rigid spores) 2. for room of 10X10 feet you can use minimum of 500g of KMnO4 + 500ml of Formaldehyde (when both are away they wont react, as soon as you mix it fumes as volcano you should no be in the room and the surroundings, you should run away from the room) 3. Seal the room to avoid the fumes to come out of the room 4. Wait for min 12 hours to act completely and efficiently 5. Wet the cloth with ammonia of 500ml and spread in the room for minimum o= f 6-7 hours to neutralize the fumes 6. Wipe the things with some disinfectant to clean and you can start your work, I promise you won=92t see any single mold for at least next 6 months = and year if you keep clean. The only disadvantage is when fumigation is going on you can=92t sit or wor= k for one day, maybe you can do this on Saturday night, So that it will be ready for Monday. This is the usual method we do in CLASS-1 rooms and CLASS-II, don=92t look at the fumes or inhale and don=92t near or it will h= arm you lot take guidance from your professor and seniors while doing this. Any thing else please feel free to contact. --=20 Yours Sincerely, *Naveen Vankadari* Lab No: N209 Graduate Student, Institute of Molecular & Cell Biology, ACADEMIA SINICA, 128 Academia Road,section-2, Nankang, Taipei-115 TAIWAN From naveenis4u from gmail.com Fri Nov 6 06:01:07 2009 From: naveenis4u from gmail.com (Naveen V) Date: Fri Nov 6 09:07:09 2009 Subject: =?iso-8859-1?q?Re=3A_Mold_problem_in_4=B0C_room?= In-Reply-To: <20091105172552.68130@gmx.net> References: <20091105172552.68130@gmx.net> Message-ID: <9cd5be270911060301m55e8733cp2dcf3db505ff774a@mail.gmail.com> Dear Thomas, The best, ideal and perfect way to abolish this problem is to fumigate your 4 degree/cold room. There are different ways to fumigate the best way is KMnO4 (Potassium permanganate) + Formaldehyde (the amount of addition will depend on size of your room) just i am illustrating the protocol 1. Vacate your room completely (the penetration and disinfection power is s= o high it can kill even rigid spores) 2. for room of 10X10 feet you can use minimum of 500g of KMnO4 + 500ml of Formaldehyde (when both are away they wont react, as soon as you mix it fumes as volcano you should no be in the room and the surroundings, you should run away from the room) 3. Seal the room to avoid the fumes to come out of the room 4. Wait for min 12 hours to act completely and efficiently 5. Wet the cloth with ammonia of 500ml and spread in the room for minimum o= f 6-7 hours to neutralize the fumes 6. Wipe the things with some disinfectant to clean and you can start your work, I promise you won=92t see any single mold for at least next 6 months = and year if you keep clean. The only disadvantage is when fumigation is going on you can=92t sit or wor= k for one day, maybe you can do this on Saturday night, So that it will be ready for Monday. This is the usual method we do in CLASS-1 rooms and CLASS-II, don=92t look at the fumes or inhale and don=92t near or it will h= arm you lot take guidance from your professor and seniors while doing this. Any thing else please feel free to contact. --=20 Yours Sincerely, *Naveen Vankadari* Lab No: N209 Graduate Student, Institute of Molecular & Cell Biology, ACADEMIA SINICA, 128 Academia Road,section-2, Nankang, Taipei-115 TAIWAN From beata.berent.maoz from gmail.com Mon Nov 9 19:11:26 2009 From: beata.berent.maoz from gmail.com (Beata Berent-Maoz) Date: Mon Nov 9 20:08:44 2009 Subject: N-acetyl cysteine treatment Message-ID: <2841ed9f0911091611h70b60f4v26b377163e01ac02@mail.gmail.com> Hi Everyone, I am trying to establish the protocol for the treatment of mice with N-acetyl cysteine (NAC) dissolved in drinking water. Does someone knows how often the NAC water has to be replaced with the fresh one (i.e how stable is NAC aqueous solution) and whether the acidic pH of the solution should be adjusted? Thank you in advance. Beata From gholamrezaahmadian from yahoo.ca Sat Nov 14 13:39:58 2009 From: gholamrezaahmadian from yahoo.ca (gholamreza ahmadian) Date: Sat Nov 14 13:46:37 2009 Subject: a queston Message-ID: <928459.98073.qm@web31915.mail.mud.yahoo.com> Dear colleagues I appreciate if anybody can help me in the following problem: I used Lpp'-ompA system (developed by Georgio et al., 1996) for surface display of a bacterial metallothioneine fused to chitin binding domain and 6Xhis-tag (on the surface of E.coli BL21). The length of this passenger protein is approximately 15kDa. We did metal binding assay using whole cell and we measured adsorption using a solution of cadmium Cd(NO3)2 in Tris-Hcl pH7.0. But it didn't show any adsorption. Hydropathy plot showed the passenger proteins are hydrophile and also it didn't show any transmembrane domain. I would like to know why does not this protein show any adsorption? I appreciate your help. Best Wishes Reza __________________________________________________________________ The new Internet Explorer? 8 - Faster, safer, easier. Optimized for Yahoo! Get it Now for Free! at http://downloads.yahoo.com/ca/internetexplorer/ From chalmers from nottingham.ac.uk Tue Nov 17 07:00:21 2009 From: chalmers from nottingham.ac.uk (Chalmers) Date: Tue Nov 17 11:43:46 2009 Subject: AKTA FPLC Mixer 925 Problem and Solution Message-ID: This is not exactly a method but could save you money. We have an AKTA FPLC with the Mixer 925 unit. We don't have a service contract so when the mixer unit stopped working and the system provided a short-circuit warning message, we asked the price of a new one. We were quoted ?10,000, which is half the price of the whole system. Presumably this is a joke, designed to force you to call in the engineer and then get a service contract, perhaps. The mixer is basically a magnetic stirrer. I opened the unit and found a little 12 V DC motor that turns the magnet. I was confused at first because it worked when I attached a 9V battery. The final explanation was that the motor had difficultly getting started, particularly at 4 C, but not so bad at room temperature. It worked intermittently for a while then failed. It had only 2 ohms resistance between the terminals (I suspect it should be 12 ohms). The ID numbers on the motor specify a custom motor supplied to the AKTA manufacturers, so you can not buy one. However, it looked suspiciously like a Premotec 990412018105. You can buy one for ?47 from RS components (the name on the motor is different but the manufacturers product code is identical). The Premotec motor is rated to run at 3600 rpm at 12 V. However, it receives only 1.89 V from the Mixer 925 unit. It therefore runs close to the specified 600 rpm when drawing power from the Mixer 925. It takes about 5 minutes to change the motor using simple tools, and we saved a significant amount of money. 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. 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 nobody from nospam.not Wed Nov 18 05:59:37 2009 From: nobody from nospam.not (Han) Date: Wed Nov 18 12:15:20 2009 Subject: AKTA FPLC Mixer 925 Problem and Solution References: Message-ID: Chalmers wrote in news:mailman.308.1258476303.1133.methods@net.bio.net: > This is not exactly a method but could save you money. > > We have an AKTA FPLC with the Mixer 925 unit. We don't have a service > contract so when the mixer unit stopped working and the system > provided a short-circuit warning message, we asked the price of a new > one. We were quoted £10,000, which is half the price of the whole > system. Presumably this is a joke, designed to force you to call in > the engineer and then get a service contract, perhaps. > > The mixer is basically a magnetic stirrer. I opened the unit and > found a little 12 V DC motor that turns the magnet. I was confused at > first because it worked when I attached a 9V battery. The final > explanation was that the motor had difficultly getting started, > particularly at 4 C, but not so bad at room temperature. It worked > intermittently for a while then failed. It had only 2 ohms resistance > between the terminals (I suspect it should be 12 ohms). > > The ID numbers on the motor specify a custom motor supplied to the > AKTA manufacturers, so you can not buy one. However, it looked > suspiciously like a Premotec 990412018105. You can buy one for £47 > from RS components (the name on the motor is different but the > manufacturers product code is identical). > > The Premotec motor is rated to run at 3600 rpm at 12 V. However, it > receives only 1.89 V from the Mixer 925 unit. It therefore runs close > to the specified 600 rpm when drawing power from the Mixer 925. > > It takes about 5 minutes to change the motor using simple tools, and > we saved a significant amount of money. > 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. > > 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. > Thanks. This is from home, and I don't know by heart whether we have that system, or another. Neither do I know whether that motor would be available in the US. I do know that I could replace the power supply on the Compaq computer that runs our HPLC after it quit. About US$38 on Ebay. -- Best regards Han email address is invalid From cjmcdermottroe from googlemail.com Wed Nov 18 11:35:18 2009 From: cjmcdermottroe from googlemail.com (Chris McDermott-Roe) Date: Wed Nov 18 12:15:26 2009 Subject: RNA ligation Message-ID: Hi everyone, I hoping to ligate a synthesised oligonucleotide to the 5' end (uncapped but not dephosphorylated) of an mRNA population. Can anybody tell me the best way of doing this? many thanks Chris From nick.theodorakis from gmail.com Wed Nov 18 20:54:25 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Wed Nov 18 21:56:23 2009 Subject: RNA ligation References: Message-ID: On Nov 18, 11:35?am, Chris McDermott-Roe wrote: > Hi everyone, > > I hoping to ligate a synthesised oligonucleotide to the 5' end (uncapped but > not dephosphorylated) of an mRNA population. ?Can anybody tell me the best > way of doing this? > > many thanks > > Chris Are they both single stranded? I think T4 RNA ligase will do that reaction. See: Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From cjmcdermottroe from googlemail.com Thu Nov 19 05:06:14 2009 From: cjmcdermottroe from googlemail.com (Chris McDermott-Roe) Date: Thu Nov 19 13:01:48 2009 Subject: RNA ligation In-Reply-To: References: Message-ID: Hi, Yes, they'll both be single stranded. One will be the mRNA that I want to tag (at the 5' end), the other will be a ss oligo. My concern is that the oligo may ligate to the 3' end of the mRNA but I guess this will depend on how I have the oligo modified? Is it sensible to assume that if it (the oligo) is 3' hydroxylated but NOT 5' phosphorylated, then it will bond with the exposed 5' phosphate on the mRNA and NOT the 3' OH?? hope this makes sense Chris 2009/11/19 Nick Theodorakis > On Nov 18, 11:35 am, Chris McDermott-Roe > wrote: > > Hi everyone, > > > > I hoping to ligate a synthesised oligonucleotide to the 5' end (uncapped > but > > not dephosphorylated) of an mRNA population. Can anybody tell me the > best > > way of doing this? > > > > many thanks > > > > Chris > > Are they both single stranded? I think T4 RNA ligase will do that > reaction. > > See: > > > Nick > > -- > Nick Theodorakis > nick_theodorakis@hotmail.com > contact form: > http://theodorakis.net/contact.html > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From Nikola.Wenta from nottingham.ac.uk Thu Nov 19 15:46:33 2009 From: Nikola.Wenta from nottingham.ac.uk (Nikola Wenta) Date: Thu Nov 19 16:29:32 2009 Subject: Sequential restriction digest Message-ID: <814A9FCB0AD790489679BFBCE6CA5E5504189AA0@VUIEXCHC.ad.nottingham.ac.uk> Dear all, I want to do a SacI/SalI digest of my plasmid in order to subclone my insert (1.2 kb) into another vector. According to NEB, the REs need different buffers, so I would have to do a sequential digest. Does anyone have a working protocol for sequential digests? Particularly I would like to know how to proceed once the first digest i.e. with SacI has linearized my original plasmid. Would you run a preparative agarose gel and cut the band out, or would you precipitate the DNA and resuspend the resulting DNA pellet in the buffer for the subsequent SalI digest? Thank you for any suggestions! 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. 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 anonwums1 from gmail.com Thu Nov 19 16:36:59 2009 From: anonwums1 from gmail.com (Adam .) Date: Thu Nov 19 17:17:22 2009 Subject: Sequential restriction digest In-Reply-To: <814A9FCB0AD790489679BFBCE6CA5E5504189AA0@VUIEXCHC.ad.nottingham.ac.uk> References: <814A9FCB0AD790489679BFBCE6CA5E5504189AA0@VUIEXCHC.ad.nottingham.ac.uk> Message-ID: <858249120911191336v4d5eafe6h1a9cf2b280f4b1de@mail.gmail.com> I generally try to avoid purification of the DNA between digests, as you lose a lot of it in the process. The way I've done sequential digest is to cut with the enzyme which uses a low salt buffer, and then spike in salt to adjust to the concentration of the second enzyme's buffer. Sometimes you have to fudge a bit and use a non-optimal buffer for the first one. An even simpler way is to use an isoschizomer for one of the enzymes so they cut in the same buffer. In the past, I've had some finicky sequential digests that worked beautifully if I just switched enzymes. I would highly recommend doing this. Adam On Thu, Nov 19, 2009 at 2:46 PM, Nikola Wenta wrote: > Dear all, > I want to do a SacI/SalI digest of my plasmid in order to subclone my > insert (1.2 kb) into another vector. According to NEB, the REs need > different buffers, so I would have to do a sequential digest. Does > anyone have a working protocol for sequential digests? Particularly I > would like to know how to proceed once the first digest i.e. with SacI > has linearized my original plasmid. Would you run a preparative agarose > gel and cut the band out, or would you precipitate the DNA and resuspend > the resulting DNA pellet in the buffer for the subsequent SalI digest? > Thank you for any suggestions! > 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. > > 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. > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From cathalgarvey from gmail.com Thu Nov 19 18:38:52 2009 From: cathalgarvey from gmail.com (Cathal Garvey) Date: Thu Nov 19 19:30:27 2009 Subject: Sequential restriction digest In-Reply-To: <814A9FCB0AD790489679BFBCE6CA5E5504189AA0@VUIEXCHC.ad.nottingham.ac.uk> References: <814A9FCB0AD790489679BFBCE6CA5E5504189AA0@VUIEXCHC.ad.nottingham.ac.uk> Message-ID: <468b1a400911191538x389a1059ydfffe225139ac66d@mail.gmail.com> I'm being forced to do likewise, with XhoI and SacI. It's a lot of frustration and sequential purification (my method of choice, as Gel Extractions are even less reliable) is my currently favoured method. However, a quick look at the Fermentas FastDigest range of enzymes shows that their speed (~15mins) is the least of their virtues; they all cut in the same buffer, which incidentally contains loading dye for gel loading as well. They're expensive compared to the regular range, but I consider the extra cost well worth it for the time saved and the satisfaction of a job done quickly. 2009/11/19 Nikola Wenta > Dear all, > I want to do a SacI/SalI digest of my plasmid in order to subclone my > insert (1.2 kb) into another vector. According to NEB, the REs need > different buffers, so I would have to do a sequential digest. Does > anyone have a working protocol for sequential digests? Particularly I > would like to know how to proceed once the first digest i.e. with SacI > has linearized my original plasmid. Would you run a preparative agarose > gel and cut the band out, or would you precipitate the DNA and resuspend > the resulting DNA pellet in the buffer for the subsequent SalI digest? > Thank you for any suggestions! > 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. > > 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. > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- letters.cunningprojects.com twitter.com/onetruecathal From mlsulliv from wisc.edu Thu Nov 19 17:35:21 2009 From: mlsulliv from wisc.edu (Michael Sullivan) Date: Thu Nov 19 19:30:34 2009 Subject: Sequential restriction digest In-Reply-To: <814A9FCB0AD790489679BFBCE6CA5E5504189AA0@VUIEXCHC.ad.nottingham.ac.uk> References: <814A9FCB0AD790489679BFBCE6CA5E5504189AA0@VUIEXCHC.ad.nottingham.ac.uk> Message-ID: <095205F3-D550-4613-96EB-68FD16CA1830@wisc.edu> For the particular combination you are talking about, both the pH and the salt concentrations of the buffers differ. What I would probably do would be to digest first with Sac I in NEB buffer 1. When you are satisfied the digest is complete (for example by running an aliquot on a gel), I would then add 1/10 volume (that is the final volume you expect with the added buffer and enzyme) NEB buffer 3 and Sal I. For this combination of enzymes, this strategy should work because both the buffer concentration and salt concentration of NEB1 are very low and will have little impact on the reaction conditions following addition of the NEB buffer 3. If you do decide to purify following the first digest, don't waste time prepping from a gel. In my mind, the only reason to gel purify is if you only want one fragment from a digest that gives multiple fragments. Precipitation is fine (and recovery is usually pretty good unless you are starting with very small amounts of DNA). You can also use a clean up type product (there are many)-- these are quick and convenient. Hope this helps. Mike On Nov 19, 2009, at 2:46 PM, Nikola Wenta wrote: > Dear all, > I want to do a SacI/SalI digest of my plasmid in order to subclone my > insert (1.2 kb) into another vector. According to NEB, the REs need > different buffers, so I would have to do a sequential digest. Does > anyone have a working protocol for sequential digests? Particularly I > would like to know how to proceed once the first digest i.e. with SacI > has linearized my original plasmid. Would you run a preparative > agarose > gel and cut the band out, or would you precipitate the DNA and > resuspend > the resulting DNA pellet in the buffer for the subsequent SalI digest? > Thank you for any suggestions! > 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. > > 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. > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods --- 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 kvolcy from mail.med.upenn.edu Thu Nov 19 20:30:36 2009 From: kvolcy from mail.med.upenn.edu (Ketna Volcy) Date: Thu Nov 19 23:41:19 2009 Subject: How long for DNA to reach nucleus? Message-ID: Did you ever get an answer for this question? -- Ketna Volcy, Ph.D. Post-doctoral Fellow University of Pennsylvania Medical Center 319 Johnson Pavilion 3610 Hamilton Walk Philadelphia, PA 19104 215-898-3846 From nick.theodorakis from gmail.com Thu Nov 19 23:23:41 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Fri Nov 20 01:08:33 2009 Subject: RNA ligation References: Message-ID: <63e4a4c6-9734-4886-baba-dab9a41e76b7@a21g2000yqc.googlegroups.com> On Nov 19, 5:06?am, Chris McDermott-Roe wrote: > Hi, > > Yes, they'll both be single stranded. ?One will be the mRNA that I want to > tag (at the 5' end), the other will be a ss oligo. ?My concern is that the > oligo may ligate to the 3' end of the mRNA but I guess this will depend on > how I have the oligo modified? ?Is it sensible to assume that if it (the > oligo) is 3' hydroxylated but NOT 5' phosphorylated, then it will bond with > the exposed 5' phosphate on the mRNA and NOT the 3' OH?? > > That sounds sensible. If the oligo has no terminal phosphates and the RNA is 5' phosphorylated, then the only reactions that should happen are ligation of the oligo to the 5'end of the RNA or circularization on the RNA. It seems to me you might be able to prevent the latter by pCp tailing the RNA first if you think that will be a problem. Disclaimer: I haven't tried any of these reactions myself. There are a couple of references mentioned in the NEB I link I posted earlier that might possibly be of use. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From aawara from pontiff-playground.org Fri Nov 20 06:11:11 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Fri Nov 20 09:13:04 2009 Subject: RNA ligation References: Message-ID: In , Chris McDermott-Roe wrote: > Hi, > > Yes, they'll both be single stranded. One will be the mRNA that I want to > tag (at the 5' end), the other will be a ss oligo. My concern is that the > oligo may ligate to the 3' end of the mRNA but I guess this will depend on > how I have the oligo modified? Is it sensible to assume that if it (the > oligo) is 3' hydroxylated but NOT 5' phosphorylated, then it will bond with > the exposed 5' phosphate on the mRNA and NOT the 3' OH?? That will NOT work. mRNAs do not have a free 5' phosphate. As you may recall, mRNAs are capped with a 7-methylguanosine cap which is covalently linked to the first transcribed nucleotide by a 5'-5' triphosphate bond. There are mammalian/eukaryotic and viral decapping enzymes, but I don't know of any that work on all mRNAs in vitro, or in vivo. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From aawara from pontiff-playground.org Fri Nov 20 06:19:26 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Fri Nov 20 09:13:11 2009 Subject: RNA ligation References: <63e4a4c6-9734-4886-baba-dab9a41e76b7@a21g2000yqc.googlegroups.com> Message-ID: <2XuNm.10633$JC2.4@newsfe06.iad> In <63e4a4c6-9734-4886-baba-dab9a41e76b7@a21g2000yqc.googlegroups.com>, Nick Theodorakis wrote: >> Yes, they'll both be single stranded. ?One will be the mRNA that I want to >> tag (at the 5' end), the other will be a ss oligo. ?My concern is that the >> oligo may ligate to the 3' end of the mRNA but I guess this will depend on >> how I have the oligo modified? ?Is it sensible to assume that if it (the >> oligo) is 3' hydroxylated but NOT 5' phosphorylated, then it will bond with >> the exposed 5' phosphate on the mRNA and NOT the 3' OH?? > > That sounds sensible. If the oligo has no terminal phosphates and the > RNA is 5' phosphorylated, then the only reactions that should happen > are ligation of the oligo to the 5'end of the RNA .. [deleted].. Won't work for mRNA - there is no free 5'phosphate. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From nick.theodorakis from gmail.com Fri Nov 20 07:47:29 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Fri Nov 20 09:13:16 2009 Subject: RNA ligation References: <63e4a4c6-9734-4886-baba-dab9a41e76b7@a21g2000yqc.googlegroups.com> <2XuNm.10633$JC2.4@newsfe06.iad> Message-ID: <4e7e0a8e-85b3-4eda-9190-7740ce3fc37e@p35g2000yqh.googlegroups.com> On Nov 20, 6:19?am, Aawara Chowdhury wrote: > In <63e4a4c6-9734-4886-baba-dab9a41e7...@a21g2000yqc.googlegroups.com>, > ?Nick Theodorakis wrote: > > >> Yes, they'll both be single stranded. ?One will be the mRNA that I want to > >> tag (at the 5' end), the other will be a ss oligo. ?My concern is that the > >> oligo may ligate to the 3' end of the mRNA but I guess this will depend on > >> how I have the oligo modified? ?Is it sensible to assume that if it (the > >> oligo) is 3' hydroxylated but NOT 5' phosphorylated, then it will bond with > >> the exposed 5' phosphate on the mRNA and NOT the 3' OH?? > > > That sounds sensible. If the oligo has no terminal phosphates and the > > RNA is 5' phosphorylated, then the only reactions that should happen > > are ligation of the oligo to the 5'end of the RNA .. [deleted].. > > Won't work for mRNA - there is no free 5'phosphate. > He specified that his RNA population is uncapped and phosphorylated in his original post. I don't know how it got that way, but I can imagine at least one scenario. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From aawara from pontiff-playground.org Fri Nov 20 21:58:13 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Sat Nov 21 16:11:12 2009 Subject: RNA ligation References: <63e4a4c6-9734-4886-baba-dab9a41e76b7@a21g2000yqc.googlegroups.com> <2XuNm.10633$JC2.4@newsfe06.iad> <4e7e0a8e-85b3-4eda-9190-7740ce3fc37e@p35g2000yqh.googlegroups.com> Message-ID: <9HINm.4826$kY2.1744@newsfe01.iad> In <4e7e0a8e-85b3-4eda-9190-7740ce3fc37e@p35g2000yqh.googlegroups.com>, Nick Theodorakis wrote: >> >> Won't work for mRNA - there is no free 5'phosphate. >> > > He specified that his RNA population is uncapped and phosphorylated in > his original post. I don't know how it got that way, but I can imagine > at least one scenario. Then it cannot be mRNA. It could be pre-mRNA or hnRNA, but mRNA needs to be capped. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From nick.theodorakis from gmail.com Fri Nov 20 22:35:57 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Sat Nov 21 16:11:18 2009 Subject: RNA ligation References: <63e4a4c6-9734-4886-baba-dab9a41e76b7@a21g2000yqc.googlegroups.com> <2XuNm.10633$JC2.4@newsfe06.iad> <4e7e0a8e-85b3-4eda-9190-7740ce3fc37e@p35g2000yqh.googlegroups.com> <9HINm.4826$kY2.1744@newsfe01.iad> Message-ID: On Nov 20, 9:58?pm, Aawara Chowdhury wrote: > In <4e7e0a8e-85b3-4eda-9190-7740ce3fc...@p35g2000yqh.googlegroups.com>, > ?Nick Theodorakis wrote: > > > > >> Won't work for mRNA - there is no free 5'phosphate. > > > He specified that his RNA population is uncapped and phosphorylated in > > his original post. I don't know how it got that way, but I can imagine > > at least one scenario. > > Then it cannot be mRNA. ?It could be pre-mRNA or hnRNA, but mRNA needs > to be capped. > Well, whatever. I'll just assume that the OP knows something about the RNA he has. But consider it could be: 1) prokaryotic or viral origin 2) mRNA that was chemically decapped or partially degraded 3) he could be looking for degradation intermediates. If you want to get nitpicky I suppose you can call 2) or 3) "mRNA- derived RNA fragments" or something, if that will make you happy. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From cjmcdermottroe from googlemail.com Sat Nov 21 19:42:34 2009 From: cjmcdermottroe from googlemail.com (Chris McDermott-Roe) Date: Sat Nov 21 23:06:28 2009 Subject: Fwd: RNA ligation In-Reply-To: References: <63e4a4c6-9734-4886-baba-dab9a41e76b7@a21g2000yqc.googlegroups.com> <2XuNm.10633$JC2.4@newsfe06.iad> <4e7e0a8e-85b3-4eda-9190-7740ce3fc37e@p35g2000yqh.googlegroups.com> <9HINm.4826$kY2.1744@newsfe01.iad> Message-ID: You're right Nick, it's chemically-decapped mRNA. You knock the 5' phosphate off with tobacco acid pyrophosphatase - it's standard to most 5' 'RACE protocols. On the question of whether its technically correct to refer to a decapped mRNA moiety as 'mRNA', I don't see any harm since an uncapped mRNA can still function as a messenger RNA in that it can generate protein products (albeit reduced in the case of cap-dependent translation but not in the case of cap-independent translation). ---------- Forwarded message -------- From: Nick Theodorakis Date: 2009/11/21 Subject: Re: RNA ligation To: methods@magpie.bio.indiana.edu On Nov 20, 9:58 pm, Aawara Chowdhury wrote: > In <4e7e0a8e-85b3-4eda-9190-7740ce3fc...@p35g2000yqh.googlegroups.com>, > Nick Theodorakis wrote: > > > > >> Won't work for mRNA - there is no free 5'phosphate. > > > He specified that his RNA population is uncapped and phosphorylated in > > his original post. I don't know how it got that way, but I can imagine > > at least one scenario. > > Then it cannot be mRNA. It could be pre-mRNA or hnRNA, but mRNA needs > to be capped. > Well, whatever. I'll just assume that the OP knows something about the RNA he has. But consider it could be: 1) prokaryotic or viral origin 2) mRNA that was chemically decapped or partially degraded 3) he could be looking for degradation intermediates. If you want to get nitpicky I suppose you can call 2) or 3) "mRNA- derived RNA fragments" or something, if that will make you happy. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From Nikola.Wenta from nottingham.ac.uk Sun Nov 22 13:53:25 2009 From: Nikola.Wenta from nottingham.ac.uk (Nikola Wenta) Date: Sun Nov 22 16:15:41 2009 Subject: Sequential restriction digest References: <200911201414.nAKEEcM06370@net.bio.net> Message-ID: <814A9FCB0AD790489679BFBCE6CA5E550418A312@VUIEXCHC.ad.nottingham.ac.uk> Dear all! Of course, using isoschizomers or hifi enzymes are an interesting idea, but are either time consuming or expensive for only one application. I have therefore tried the low-salt-buffer-first suggestion, and it really worked great! Complete digest, no waste of agarose and extraction kits, simply two-step-single-tube ;-) Thank you all very much for your suggestions! 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. 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 mskeels from stlawu.edu Mon Nov 23 18:32:11 2009 From: mskeels from stlawu.edu (Matthew Skeels) Date: Mon Nov 23 19:15:38 2009 Subject: Looking for Hot Bonnet for PTC150 Message-ID: <4B0B1B7B.4060106@stlawu.edu> Hello, I was wondering if someone could help me find a hot bonnet for a MJR PTC150? [used or new?] Cheers, MS From blackhole from abuse.plus.com Tue Nov 24 11:20:52 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Tue Nov 24 12:19:18 2009 Subject: Looking for Hot Bonnet for PTC150 References: Message-ID: Historians believe that in newspost on Mon, 23 Nov 2009, Matthew Skeels penned the following literary masterpiece: >I was wondering if someone could help me find a hot bonnet for a MJR >PTC150? [used or new?] You maybe surprised but Ebay is worth a look! Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From editor from gene-quantification.info Tue Nov 24 07:47:10 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Tue Nov 24 12:19:28 2009 Subject: qPCR 2010 Event - Call for Abstracts Message-ID: qPCR 2010 Event - Call for Abstracts www.qPCR2010-Vienna.net ----------------------------------------------------- BioEPS GmbH is organizing the qPCR 2010 Event taking place April 7th =96 9th, 2010 in Vienna, Austria. Scientists from all around the world will come to exchange ideas, share experiences, and discuss the exciting future of the perhaps most powerful analytical technology ever developed in the life sciences area =96 the quantitative real-time polymerase chain reaction (qPCR). We have the pleasure to announce the Call for Abstracts for the qPCR 2010 Event ! Finally we want to present 40 scientific talks from international scientists and diagnostic companies in the qPCR field who will show their latest research findings and newest technologies. The focus of the qPCR 2010 Event will be =93The ongoing evolution of qPCR=94 representing all new and emerging techniques, applications and data analysis methods. Deadline is 31st January 2010 =3D> http://submission.qPCR2010-Vienna.ne= t Talk topics: -------------- MIQE and QM strategies in qPCR The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results. QM strategies in real- time PCR to guarantee better and more valid results. Keynote speaker: Prof. Stephen Bustin High throughput quantitative PCR =96 digital PCR 384 well applications, new high throughput platforms, droplet PCR, qPCR robotics, digital PCR, gene expression real-time RT-PCR arrays (mRNA and microRNA), quantitative multiplexing, =85 Keynote speaker: Prof. Mikael Kubista, Dr. Philip Day, Dr. Ken Livak HRM =96 High Resolution Melting SNP analysis, HRM =3D high resolution melt applications, Epigenetics, methylation markers, HRM platform comparison, etc =85 Keynote Speaker Prof. Carl Wittwer, Prof. Claudio Orlando Circulating nucleic acids Analysis of circulating RNAs and DNA and microRNAs as diagnostic and prognostic marker, =85 Speaker: Dr. Pamela Pinzani, Dr. Alfred Sch=F6ller, Dr. Jim Huggett Single=96cell qPCR single-cell sampling, pre-amplification techniques, laser micro- dissection, sub-cellular PCR, micro-manipulation of cell clusters, cellular micro injection, FACS spotting, single cell handling, pre- amplification, =85 Keynote speaker: Dr. Michael W. Pfaffl, Dr. Anders Stahlberg RNAi =96 microRNA =96 siRNA Applications =96 miRNA normalisation RNAi mechanism, microRNA extraction, qRT-PCR technologies to detect microRNA, microRNA normalisation strategies, siRNA applications in combination with qRT-PCR, microRNA targets and microRNA precursors, new siRNA manipulation and microRNA technologies, Keynote speaker: Prof. Jo Vandesompele, Dr. Mirco Castoldi qPCR BioStatistics & BioInformatics software applications, data mining, calculation of relative expression, primer and probe design on mRNA and microRNA level, real- time PCR efficiency determination, mathematical modelling, multivariate expression profiling, statistics in real-time PCR, data management, multiway expression profiling, multiple regression analysis, 3D data visualization, ... Speaker: Dr. Ales Tichopad, Dr. Jan Hellemans, Dr. Anders Bergkvist An online registration and abstract submission software CONFTOOL is available here =3D> http://registration.qPCR2010-Vienna.net ----------------------------- About qPCR: Using qPCR the amount of target nucleic acid in a complex sample can be determined with high precision, great accuracy, excellent specificity and the ultimate sensitivity of detecting a single molecule. The technique has revolutionized all molecular sciences and diagnostic applications. Conference presentations will include MIQE guidelines & QM strategies in qPCR, high performance nucleic acid extraction, single-cell applications, Epigenetics & High-Resolution- Melt analysis, circulating nucleic acids, and application involving RNAi and microRNA. Further developments of qPCR technology will be presented include improved instrumentation, miniaturization, high throughput platforms, cost efficacy, validity, flexibility, quality assessment and reliable Cq calculations, expression data comparisons, and interpretation. Today there is no field in the life sciences research, molecular biology and diagnostics areas that has not introduced qPCR technology for nucleic acid analysis. The combination with reverse transcription enables determination of mRNA, microRNA and widely opens the window for =93Transcriptomics=94 =96 the first step of quantitative =93Gene Expression Profiling=94 and =93Functional Genomics=94. An Industrial Exhibition will take place parallel to the symposium, with 32 leading biotechnology companies presenting their latest developments, including real-time PCR cyclers, NA extraction robots, consumables, new fluorescence dyes, NA detection and amplification chemistries, as well as real-time PCR data analysis software. For more information about the qPCR 2010 event contact Dr. Martina Reiter Martina.Reiter@bioeps.com Hope to meet you in April in Vienna! Michael Pfaffl From tk from shaggy.csail.mit.edu Tue Nov 24 09:16:30 2009 From: tk from shaggy.csail.mit.edu (Tom Knight) Date: Tue Nov 24 12:19:34 2009 Subject: Looking for Hot Bonnet for PTC150 References: Message-ID: Matthew Skeels writes: > I was wondering if someone could help me find a hot bonnet for a MJR > PTC150? [used or new?] Bio-rad now maintains MJ Research instruments; I believe they will have this part. From coatej from gmail.com Tue Nov 24 08:46:40 2009 From: coatej from gmail.com (Jeremy Coate) Date: Tue Nov 24 12:19:39 2009 Subject: lyophilizing leaf tissue Message-ID: <42f327750911240546r2ab05ba6l2490a3ccd86ea543@mail.gmail.com> Hi savy methodologists, I need to lyophilize leaf tissue prior to shipping overseas. The tissue wil= l be used for analysis of lipids, which are vulnerable to endogenous lipases, so I am planning to collect the tissue into liquid N2, freeze dry and ship on dry ice to try to ensure that these lipases don't have a chance to do their thing. I have never used a lyophilizer and have concerns that I'm using it incorrectly, and that the samples are thawing. I'm using a Labconco FreeZone 18 freeze dryer with a bulk dryer unit on top=From haleyl from u.washington.edu Tue Nov 24 17:12:50 2009 From: haleyl from u.washington.edu (Haley Lindsey) Date: Tue Nov 24 19:45:04 2009 Subject: Purifying 5' phosphorylated primers for mutagenesis Message-ID: <637ccded0911241412y6b11a7bdhace8c042a26d993@mail.gmail.com> I'm doing some site-directed mutagenesis with a kit from NEB. It is recommended in the kit directions that the 5' phosphorylated primers used for the reaction are purified either using RP-HPLC or PAGE. My issue is that these purification methods are *really* expensive. (I've been getting my primers through Invitrogen, but I've also looked at a few other places and it's the same story.) Has anyone had success using primers purified another (less expensive) way or done the purification in house? Thank you, Haley, University of Washington From lautys from gmail.com Tue Nov 24 22:13:07 2009 From: lautys from gmail.com (lautys) Date: Tue Nov 24 22:20:10 2009 Subject: lyophilizing leaf tissue In-Reply-To: <42f327750911240546r2ab05ba6l2490a3ccd86ea543@mail.gmail.com> References: <42f327750911240546r2ab05ba6l2490a3ccd86ea543@mail.gmail.com> Message-ID: Hi Jeremy, Lyophilizers suppose to fully dry up water content of the sample, so "thawing" shouldn't be the concern. lautys On Tue, Nov 24, 2009 at 9:46 PM, Jeremy Coate wrote: > Hi savy methodologists, > > I need to lyophilize leaf tissue prior to shipping overseas. The tissue > will > be used for analysis of lipids, which are vulnerable to endogenous lipases, > so I am planning to collect the tissue into liquid N2, freeze dry and ship > on dry ice to try to ensure that these lipases don't have a chance to do > their thing. I have never used a lyophilizer and have concerns that I'm > using it incorrectly, and that the samples are thawing. > > I'm using a Labconco FreeZone 18 freeze dryer with a bulk dryer unit on > top_______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods >