From pow.joshi from gmail.com Sun Dec 2 16:20:14 2007 From: pow.joshi from gmail.com (Pow Joshi) Date: Mon Dec 3 01:11:13 2007 Subject: Methods Digest, Vol 30, Issue 20 In-Reply-To: <440923e10711301754i60d94e4bk8cf4f515bcfef537@mail.gmail.com> References: <200711301703.lAUH3lY12228@net.bio.net> <440923e10711301754i60d94e4bk8cf4f515bcfef537@mail.gmail.com> Message-ID: <710764ea0712021320h383f9d6cgdd4fc4e274d2bdba@mail.gmail.com> On 30/11/2007, doaa zineldeen wrote: > > hello > I am having problem in detection of my protein which is a 72 KD kinase by > western blotting > I am using a commercial one , it could detect my kinase by IF howevere on > western blotting many faint bands are seen and high background > I tried different blocking and different dilutions of the primary and > secondary and increasedwashing time but no clear band > anyone can help would you please let us know whether you see greater number of bands lower to 72kDa or higher? Also, if it is a comercial Ab, they do have a data sheet that gives you a complete characterization of the Ab...you could check and see if they do use it for westerns, and what their recommendations are....(if you havent already done so) ... further, is your kinase also autophosphorylated? in which case, would you be using phospho specific epitope? best, Pow thank you > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From rosaak from gmail.com Tue Dec 4 04:57:55 2007 From: rosaak from gmail.com (rosaak) Date: Tue Dec 4 11:49:18 2007 Subject: Transgenic mice vector construct Message-ID: <69f53a6d-ea41-41af-b307-1ad760fe2ffc@b40g2000prf.googlegroups.com> Hi all, I have constructed a vector consting cDNA for making transgenic mice. The backbone is pCI contining CMV promoter.For microinjections i need to linearise the vector., and there are no suitable sites for it. My doubt is that can i use a set of primers to pcr amplify CMVpromoter- cDNA-polyA region from the construct and use it for microinjection. [i'll be using a highfidility enzyme for pcr.] Is it techically ok to do this.If so how will i purfy it ? Please reply regards, RP From pow.joshi from gmail.com Tue Dec 4 14:51:58 2007 From: pow.joshi from gmail.com (Pow Joshi) Date: Tue Dec 4 15:01:43 2007 Subject: Transgenic mice vector construct In-Reply-To: <69f53a6d-ea41-41af-b307-1ad760fe2ffc@b40g2000prf.googlegroups.com> References: <69f53a6d-ea41-41af-b307-1ad760fe2ffc@b40g2000prf.googlegroups.com> Message-ID: <710764ea0712041151g593c6279mabd45f6c9a9e04dd@mail.gmail.com> On 04/12/2007, rosaak wrote: > > Hi all, > > I have constructed a vector consting cDNA for making transgenic mice. > The backbone is pCI contining CMV promoter.For microinjections i need > to linearise the vector., and there are no suitable sites for it. > > My doubt is that can i use a set of primers to pcr amplify CMVpromoter- > cDNA-polyA region from the construct and use it for microinjection. > [i'll be using a highfidility enzyme for pcr.] > > Is it techically ok to do this.If so how will i purfy it ? well, I cannot say anything about the pcr, for purification, I use the Qiagen kit, and it works quite well, at least for my purposes.... but then Iuse zebrafish eggs. You could also use the standard phenol chloroform extraction foillowed ny isopropanol/ ethanol precipitation. Hope that helps Pow Please reply > > regards, > > RP > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From R.Jayakumar from roswellpark.org Tue Dec 4 15:09:19 2007 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Tue Dec 4 17:39:45 2007 Subject: Transgenic mice vector construct In-Reply-To: <710764ea0712041151g593c6279mabd45f6c9a9e04dd@mail.gmail.com> Message-ID: <97101976F8A044468CA74FE11883B90E173E793B@VISTA.roswellpark.org> I would not do PCR of a construct to use for microinjection, unless you are sure the PCR did not introduce any artifactual mutations in the construct especially the promoter and cDNA sequence. Alternatively, you could point mutate the construct at the location where you want to linearise to introduce a restriction site of your interest and then linearise it. Hope that helps. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Pow Joshi Sent: Tuesday, December 04, 2007 2:52 PM To: rosaak Cc: methods@magpie.bio.indiana.edu Subject: Re: Transgenic mice vector construct On 04/12/2007, rosaak wrote: > > Hi all, > > I have constructed a vector consting cDNA for making transgenic mice. > The backbone is pCI contining CMV promoter.For microinjections i need > to linearise the vector., and there are no suitable sites for it. > > My doubt is that can i use a set of primers to pcr amplify > CMVpromoter- cDNA-polyA region from the construct and use it for microinjection. > [i'll be using a highfidility enzyme for pcr.] > > Is it techically ok to do this.If so how will i purfy it ? well, I cannot say anything about the pcr, for purification, I use the Qiagen kit, and it works quite well, at least for my purposes.... but then Iuse zebrafish eggs. You could also use the standard phenol chloroform extraction foillowed ny isopropanol/ ethanol precipitation. Hope that helps Pow Please reply > > regards, > > RP > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ 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 dagrg_spamout from online.no Fri Dec 7 10:12:39 2007 From: dagrg_spamout from online.no (Dag Rune Gjellesvik) Date: Fri Dec 7 13:53:25 2007 Subject: speI xbaI sublconing References: Message-ID: <13lionhkg31cn1f@corp.supernews.com> "V Arunachalam" skrev i melding news:mailman.782.1196360538.23109.methods@net.bio.net... > Hi, > I am facing problems in cohesive subcloning a 500 bp insert from the > double digest of SpeI and XbaI onto a vector of 13.5 Kb size at the same > sites. Please suggest me is it correct strategy as these two enzymes > generate similar ends though differ in recognition sites. > > > > ____________________________________________________________________________________ > Never miss a thing. Make Yahoo your home page. > http://www.yahoo.com/r/hs Hello, I understand - you cannot dephosphorylate both vector and insert, and both may circularize since they have compatible ends. I have been in the same situation. I suggest that if you can cut your vector with only one of the enzymes, lets say SphI, and dephosphorylate your insert. If you do the ligation in the presence of SphI restriction enzyme, you will prevent your vector from circularizing. However, the XbaI end of your insert may ligate to either end of the SphI cut vector. After a period for ligation, inactivate the SphI enzyme by heat treatment, and add more ligase to allow your construct to close (circularize). You may have to screen for correctly oriented inserts, if that matters. I hope this will work, Dag Rune Gjellesvik From tk from csail.mit.edu Fri Dec 7 16:40:34 2007 From: tk from csail.mit.edu (Tom Knight) Date: Fri Dec 7 18:28:42 2007 Subject: speI xbaI sublconing References: <13lionhkg31cn1f@corp.supernews.com> Message-ID: "V Arunachalam" skrev i melding news:mailman.782.1196360538.23109.methods@net.bio.net... > I am facing problems in cohesive subcloning a 500 bp insert from the > double digest of SpeI and XbaI onto a vector of 13.5 Kb size at the same > sites. Please suggest me is it correct strategy as these two enzymes > generate similar ends though differ in recognition sites. Two options: 1) Ignore the problem and select one of the two orientations after the fact. About half should be in the orientation you want. 2) Add SpeI or XbaI to the ligation mix. This will force the ligation which recreates the SpeI or XbaI site to be cut, favoring the ligation of the vector SpeI site with the insert XbaI site, and vice-versa. From mprigge from indiana.edu Fri Dec 7 20:19:17 2007 From: mprigge from indiana.edu (Michael J. Prigge) Date: Fri Dec 7 23:00:05 2007 Subject: speI xbaI sublconing Message-ID: <142F4A8C-4B8F-48EC-A1C9-15575C8CD418@indiana.edu> You didn't mention any problems with the digestion, but I thought I would throw in this reminder, just in case. XbaI is susceptible to dam methylation, so TCTAGAtc and gaTCTAGA will not be cut. If this isn't the problem, I would try Tom's options, possibly screening colonies by PCR with an insert primer and a vector primer for the correct orientation. Good Luck, Mike >> "V Arunachalam" skrev i melding >> news:mailman.782.1196360538.23109.methods from net.bio.net... >> > I am facing problems in cohesive subcloning a 500 bp insert from >> the >> > double digest of SpeI and XbaI onto a vector of 13.5 Kb size at >> the same >> > sites. Please suggest me is it correct strategy as these two >> enzymes >> > generate similar ends though differ in recognition sites. >> > Tom Knight via methods%40net.bio.net (by tk from csail.mit.edu) > Two options: > 1) Ignore the problem and select one of the two orientations after the > fact. About half should be in the orientation you want. > 2) Add SpeI or XbaI to the ligation mix. This will force the ligation > which recreates the SpeI or XbaI site to be cut, favoring the > ligation of the vector SpeI site with the insert XbaI site, and > vice-versa. > > From aawara from FEMA-trailer.org Fri Dec 7 22:52:15 2007 From: aawara from FEMA-trailer.org (Aawara Chowdhury) Date: Fri Dec 7 23:26:18 2007 Subject: speI xbaI sublconing References: <13lionhkg31cn1f@corp.supernews.com> Message-ID: In , Tom Knight wrote: > "V Arunachalam" skrev i melding > news:mailman.782.1196360538.23109.methods@net.bio.net... >> I am facing problems in cohesive subcloning a 500 bp insert from the >> double digest of SpeI and XbaI onto a vector of 13.5 Kb size at the same >> sites. Please suggest me is it correct strategy as these two enzymes >> generate similar ends though differ in recognition sites. > > Two options: > 1) Ignore the problem and select one of the two orientations after the > fact. About half should be in the orientation you want. > 2) Add SpeI or XbaI to the ligation mix. This will force the ligation > which recreates the SpeI or XbaI site to be cut, favoring the > ligation of the vector SpeI site with the insert XbaI site, and > vice-versa. SpeI and XbaI have compatible overhangs, and uni-molecular reactions are always favored over bi-molecular reactions. Unless the original poster dephosphorylates the ends of his cut vector, it will recircularize very efficiently, and none of his clones will contain the insert. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From David.L.Haviland from uth.tmc.edu Sat Dec 8 11:23:17 2007 From: David.L.Haviland from uth.tmc.edu (Haviland, David L) Date: Sat Dec 8 17:27:11 2007 Subject: speI xbaI sublconing References: <142F4A8C-4B8F-48EC-A1C9-15575C8CD418@indiana.edu> Message-ID: <76E50A283589324FA6A1999EEFBB1341011A17CB@UTHEVS1.mail.uthouston.edu> Mike: I won't argue with you but in my limited experience with XbaI and using various vectors in HB101, XL-1 blues, Top 10F's, and SURE cells, what problems I had in ligation were never chased to XbaI methylation. I dont' doubt what you are saying and perhaps in other bugs it would make a difference. To me the compatible overhangs are what dogs this ligation, and I'd reach for the SAP in a heartbeat. David -----Original Message----- From: methods-bounces@oat.bio.indiana.edu on behalf of Michael J. Prigge Sent: Fri 12/7/2007 7:19 PM To: methods@magpie.bio.indiana.edu Subject: speI xbaI sublconing You didn't mention any problems with the digestion, but I thought I would throw in this reminder, just in case. XbaI is susceptible to dam methylation, so TCTAGAtc and gaTCTAGA will not be cut. If this isn't the problem, I would try Tom's options, possibly screening colonies by PCR with an insert primer and a vector primer for the correct orientation. Good Luck, Mike >> "V Arunachalam" skrev i melding >> news:mailman.782.1196360538.23109.methods from net.bio.net... >> > I am facing problems in cohesive subcloning a 500 bp insert from >> the >> > double digest of SpeI and XbaI onto a vector of 13.5 Kb size at >> the same >> > sites. Please suggest me is it correct strategy as these two >> enzymes >> > generate similar ends though differ in recognition sites. >> > Tom Knight via methods%40net.bio.net (by tk from csail.mit.edu) > Two options: > 1) Ignore the problem and select one of the two orientations after the > fact. About half should be in the orientation you want. > 2) Add SpeI or XbaI to the ligation mix. This will force the ligation > which recreates the SpeI or XbaI site to be cut, favoring the > ligation of the vector SpeI site with the insert XbaI site, and > vice-versa. > > _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From josenet from tiscali.co.uk Sat Dec 8 09:42:42 2007 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Sat Dec 8 17:27:17 2007 Subject: speI xbaI sublconing References: <13lionhkg31cn1f@corp.supernews.com> Message-ID: <5rvp5sF16sadpU1@mid.individual.net> "DK" wrote in message news:iFo6j.17$2R3.11@newsfe02.lga... > aawara@FEMA-trailer.org wrote: >>In , >> Tom Knight wrote: >> >>> "V Arunachalam" skrev i melding >>> news:mailman.782.1196360538.23109.methods@net.bio.net... >>>> I am facing problems in cohesive subcloning a 500 bp insert from the >>>> double digest of SpeI and XbaI onto a vector of 13.5 Kb size at the >>>> same >>>> sites. Please suggest me is it correct strategy as these two enzymes >>>> generate similar ends though differ in recognition sites. >>> >>> Two options: >>> 1) Ignore the problem and select one of the two orientations after the >>> fact. About half should be in the orientation you want. >>> 2) Add SpeI or XbaI to the ligation mix. This will force the ligation >>> which recreates the SpeI or XbaI site to be cut, favoring the >>> ligation of the vector SpeI site with the insert XbaI site, and >>> vice-versa. >> >>SpeI and XbaI have compatible overhangs, and uni-molecular reactions are >>always favored over bi-molecular reactions. >> >>Unless the original poster dephosphorylates the ends of his cut vector, >>it will recircularize very efficiently, and none of his clones will >>contain >>the insert. > > The situation is no different from blunt cloning. Easy to deal with by > throwing a large excess of the insert. 20X or more. With everything > done properly, a reasonable percent of clones will contain an insert. > > DK I'd agree with that and go for approach (1) above. Minipreps are easy and quick to do for screening, and usually the first handful give you at least one of the constructs you're looking for... I wouldn't worry too much, just mix the right components with a large excess of insert and screen for the correct one. Jose -- Musha ring dum a doo dum a dah - www.mcnach.com Current fave guitar: Fender 'Sambora' Stratocaster Current fave bass: Tanglewood Warrior III Fender Stratocaster - part coffee table, part spaceship. From pjie2 from cam.ac.uk Sat Dec 8 10:01:59 2007 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Sat Dec 8 17:27:21 2007 Subject: speI xbaI sublconing References: <13lionhkg31cn1f@corp.supernews.com> Message-ID: <5rvpv8F16rrurU1@mid.individual.net> DK wrote: > aawara@FEMA-trailer.org wrote: >> >> Unless the original poster dephosphorylates the ends of his cut >> vector, >> it will recircularize very efficiently, and none of his clones will >> contain the insert. > > The situation is no different from blunt cloning. Easy to deal with by > throwing a large excess of the insert. 20X or more. With everything > done properly, a reasonable percent of clones will contain an insert. Blunt cloning is *also* much easier if you dephosphorylate the vector. Peter From mlsulliv from wisc.edu Sun Dec 9 13:10:31 2007 From: mlsulliv from wisc.edu (Michael Sullivan) Date: Sun Dec 9 14:42:23 2007 Subject: speI xbaI sublconing (dam methylation) In-Reply-To: <76E50A283589324FA6A1999EEFBB1341011A17CB@UTHEVS1.mail.uthouston.edu> References: <142F4A8C-4B8F-48EC-A1C9-15575C8CD418@indiana.edu> <76E50A283589324FA6A1999EEFBB1341011A17CB@UTHEVS1.mail.uthouston.edu> Message-ID: I DID have this (i.e. overlapping dam methylation) "problem" with XbaI. Of course it was totally my fault: not paying enough attention when I was designing primers. I couldn't understanding why I wasn't recovering clones for such a simple ligation... then I noticed that following digestion, the "vector" band was too big. When I looked more carefully, I realized I had designed my XbaI site with overlapping dam methylation. Every single thing I had prepped had the insert-- it's just digesting the plasmids (prepped out of XL-1 Blue) with XbaI would not release the insert! Needless to say, I've been pretty careful about this issue from then on! Mike On Dec 8, 2007, at 10:23 AM, Haviland, David L wrote: > Mike: > > I won't argue with you but in my limited experience with XbaI and > using various vectors in HB101, XL-1 blues, Top 10F's, and SURE > cells, what problems I had in ligation were never chased to XbaI > methylation. I dont' doubt what you are saying and perhaps in > other bugs it would make a difference. > > To me the compatible overhangs are what dogs this ligation, and I'd > reach for the SAP in a heartbeat. > > David > > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu on behalf of Michael J. > Prigge > Sent: Fri 12/7/2007 7:19 PM > To: methods@magpie.bio.indiana.edu > Subject: speI xbaI sublconing > > You didn't mention any problems with the digestion, but I thought I > would throw in this reminder, just in case. XbaI is susceptible to > dam methylation, so TCTAGAtc and gaTCTAGA will not be cut. If this > isn't the problem, I would try Tom's options, possibly screening > colonies by PCR with an insert primer and a vector primer for the > correct orientation. > Good Luck, > Mike > >>> "V Arunachalam" skrev i melding >>> news:mailman.782.1196360538.23109.methods from net.bio.net... >>>> I am facing problems in cohesive subcloning a 500 bp insert from >>> the >>>> double digest of SpeI and XbaI onto a vector of 13.5 Kb size at >>> the same >>>> sites. Please suggest me is it correct strategy as these two >>> enzymes >>>> generate similar ends though differ in recognition sites. >>> >> Tom Knight via methods%40net.bio.net (by tk from csail.mit.edu) >> Two options: >> 1) Ignore the problem and select one of the two orientations after >> the >> fact. About half should be in the orientation you want. >> 2) Add SpeI or XbaI to the ligation mix. This will force the >> ligation >> which recreates the SpeI or XbaI site to be cut, favoring the >> ligation of the vector SpeI site with the insert XbaI site, and >> vice-versa. >> >> > > > > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > > _______________________________________________ > 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 jrsimkhada from gmail.com Mon Dec 10 06:28:33 2007 From: jrsimkhada from gmail.com (jrsimkhada@gmail.com) Date: Mon Dec 10 13:22:57 2007 Subject: how to calculate Kcat value of enzyme Message-ID: <37197b5c-9d53-4560-acb3-2abc2f313ca2@a35g2000prf.googlegroups.com> Good evening molecular weight of enzyme is 60 kDa and I took 4 ug of it in each reaction. Vmax and km values were obtained as 1.24 mmole/min/mg protein and mM. so how could i calculate Kcat value of the enzyme ? I will be very grateful for your kind cooperation. From laht0028 from umn.edu Mon Dec 10 13:12:44 2007 From: laht0028 from umn.edu (Lahti) Date: Mon Dec 10 13:23:11 2007 Subject: Looking for the optimal Cell Lysis Buffer Message-ID: <14258171.post@talk.nabble.com> I am looking for a cell lysis buffer that will help me solubilize a lysosmal cathepsin. I am currently finding the Cathepsin in my cell lysate samples as well as my cell pellet. The cathepsin may be membrane associated (with the plasma membrane or other cellular membranes). What are detergents and/or buffers that might best aid me in removing a cathepsin from my cellular debris? Lahti -- View this message in context: http://www.nabble.com/Looking-for-the-optimal-Cell-Lysis-Buffer-tp14258171p14258171.html Sent from the Bio.net - Methods mailing list archive at Nabble.com. From edward.weiss from utoronto.ca Tue Dec 11 15:51:35 2007 From: edward.weiss from utoronto.ca (Edward Weiss) Date: Tue Dec 11 23:41:08 2007 Subject: Rhodamine dUTP and capillary eletrophoresis Message-ID: <475EF857.5020108@utoronto.ca> Hi all, Just wondering if anyone's run into this problem or has any suggestions for how to remedy it. We've started labeling select PCR products with rhodamine-dUTP and running them on our ABI 3730xl. So far, so good, at least when the fragments are run right after the PCR. However, we've found that if we try using a restriction enzyme or a mismatch-recognizing enzyme (CEL I) on these labeled samples, the intensity on the ABI is so weak as to be almost negligible. These same samples look fine when run on an agarose gel, so I'm guessing that for some reason the labeled products are not making it into the capillaries, perhaps to the rhodamine binding something or another. Any ideas about how we can circumvent this? Thanks, Ed -- Edward S. Weiss Research Technologist Genetic Analysis Facility The Centre for Applied Genomics @ The Hospital for Sick Children Room 14-601S, East Tower 101 College Street Toronto, ON M5G 1L7 Canada 416-813-8642 edward.weiss@utoronto.ca From gtaccioli from gmail.com Wed Dec 12 00:13:57 2007 From: gtaccioli from gmail.com (gtaccioli@gmail.com) Date: Wed Dec 12 15:38:54 2007 Subject: ADCC protocol needed Message-ID: <587aba3f-7eda-4706-bba9-001cfe638d25@t1g2000pra.googlegroups.com> Dear Netters: I would greatly appreciate sharing a protocol for ADCC (Ab mediated cytotoxicity), non-radioactive preferred. I am sorry to post this here but looks like the immunology.bionet is collapsed. Thank in advance for your help Guillermo From novalidaddress from nurfuerspam.de Wed Dec 12 03:07:27 2007 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Dec 12 15:39:00 2007 Subject: Rhodamine dUTP and capillary eletrophoresis References: Message-ID: Dear Ed, have you tried desalting or doing a (PCR) cleanup before you analyze the samples? Best regards, Wolfgang From ivanoov from gmail.com Wed Dec 12 05:56:59 2007 From: ivanoov from gmail.com (chovek69) Date: Wed Dec 12 15:39:13 2007 Subject: Rhodamine dUTP and capillary eletrophoresis References: Message-ID: <27ce64f6-7b8d-4cb3-93a0-e6f615bad59c@a35g2000prf.googlegroups.com> Yeah Wolfgang makes a good point. Capillary electrophoresis is very sensitive to salt concentrations above the regular PCR ones (e.g in restrictase buffers). Try desalting or diluting samples 1/5 or 1/10 with ddH2O before separation. Hope this will help On Dec 12, 10:07 am, WS wrote: > Dear Ed, > > have you tried desalting or doing a (PCR) cleanup before you analyze > the samples? > > Best regards, > > Wolfgang From edward.weiss from utoronto.ca Wed Dec 12 15:50:55 2007 From: edward.weiss from utoronto.ca (Edward Weiss) Date: Wed Dec 12 17:45:48 2007 Subject: Rhodamine dUTP and capillary eletrophoresis In-Reply-To: <27ce64f6-7b8d-4cb3-93a0-e6f615bad59c@a35g2000prf.googlegroups.com> References: <27ce64f6-7b8d-4cb3-93a0-e6f615bad59c@a35g2000prf.googlegroups.com> Message-ID: <476049AF.6090705@utoronto.ca> I don't think the salt concentration is a particular issue -- we've done exactly the same procedure using labeled primers instead of labeled dUTP, and it works fine. I don't imagine the salt concentration of the dUTP stock would be so high as to make things significantly different in the final product. I've tried 1/10 and 1/20 dilutions too, with no luck. Thanks for the help, Ed chovek69 wrote: > Yeah Wolfgang makes a good point. Capillary electrophoresis is very > sensitive to salt concentrations above the regular PCR ones (e.g in > restrictase buffers). Try desalting or diluting samples 1/5 or 1/10 > with ddH2O before separation. > > Hope this will help > > On Dec 12, 10:07 am, WS wrote: > >> Dear Ed, >> >> have you tried desalting or doing a (PCR) cleanup before you analyze >> the samples? >> >> Best regards, >> >> Wolfgang >> > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Edward S. Weiss Research Technologist Genetic Analysis Facility The Centre for Applied Genomics @ The Hospital for Sick Children Room 14-601S, East Tower 101 College Street Toronto, ON M5G 1L7 Canada 416-813-8642 edward.weiss@utoronto.ca From drpragnyadas from gmail.com Fri Dec 14 14:06:14 2007 From: drpragnyadas from gmail.com (Pragnya Das) Date: Fri Dec 14 15:59:43 2007 Subject: Rhodamine dUTP and capillary eletrophoresis In-Reply-To: References: Message-ID: Hi everyone I'm facing a serious cloning issue. I'm trying to clone a gene into an RNAi vector (pRFP ~6kb), by PCR and then subclone it into RCAS (~11kb) vector. Even after cutting out the insert from RFP, followed by gel purification, i'm still getting the insert cloned into RFP, although I ligate my insert in RCAS. Why is this happening? has anyone faced a problem like this before? Pragnya Das From Glen.Alberts from vai.org Fri Dec 14 11:07:58 2007 From: Glen.Alberts from vai.org (Alberts, Glen) Date: Fri Dec 14 16:00:25 2007 Subject: Red/ET recombination Message-ID: <9A6509A94A33124187515A9901DC551F022783FB0F@VAIEXCH05.vai.org> Did anyone in your lab have success with the Gene Bridges Red/ET kit? Thanks, Glen - VAI- Grand Rapids, MI This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you. From josenet from tiscali.co.uk Fri Dec 14 18:51:47 2007 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Fri Dec 14 22:14:26 2007 Subject: Rhodamine dUTP and capillary eletrophoresis References: Message-ID: <5sgj91F19armfU1@mid.individual.net> "Pragnya Das" wrote in message news:mailman.942.1197666010.23109.methods@net.bio.net... > Hi everyone > I'm facing a serious cloning issue. > > I'm trying to clone a gene into an RNAi vector (pRFP ~6kb), by PCR and > then > subclone it into RCAS (~11kb) vector. Even after cutting out the insert > from > RFP, followed by gel purification, i'm still getting the insert cloned > into > RFP, although I ligate my insert in RCAS. Why is this happening? has > anyone > faced a problem like this before? > > Pragnya Das I think it's more likely that you you are not recloning into pRFP, but rather you are picking up background clones derived from a few molecules of the original plasmid that was not cut in the first place. Even if you gel purify there will always be a few other fragments carried over, as well as the band you are cutting out. Are you recovering many or just a few colonies? How big is the insert? If you're only picking up these, it suggests a severe problem with your cloning. I assume your digest looks good. Now wanting to insult you... but I've done this myself: are you sure you are using the correct antibiotic? if both plasmids use a different selection, and you're inadvertently plating your ligations with the antibiotic needed for pRFP, then you'd get a result like you're describing. If that is ok, are you sure your ligation has worked? Do you have a positive control? Jose From josenet from tiscali.co.uk Sat Dec 15 07:17:30 2007 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Sat Dec 15 12:31:04 2007 Subject: Cloning problems Message-ID: <5shuv2F1982j1U1@mid.individual.net> Whops!: When starting a new thread... do that, don't simply reply to an old message and leave an irrelevant subject title behind! I replied without realising, so here it is amended: "Pragnya Das" wrote in message news:mailman.942.1197666010.23109.methods@net.bio.net... > Hi everyone > I'm facing a serious cloning issue. > > I'm trying to clone a gene into an RNAi vector (pRFP ~6kb), by PCR and > then > subclone it into RCAS (~11kb) vector. Even after cutting out the insert > from > RFP, followed by gel purification, i'm still getting the insert cloned > into > RFP, although I ligate my insert in RCAS. Why is this happening? has > anyone > faced a problem like this before? > > Pragnya Das I think it's more likely that you you are not recloning into pRFP, but rather you are picking up background clones derived from a few molecules of the original plasmid that was not cut in the first place. Even if you gel purify there will always be a few other fragments carried over, as well as the band you are cutting out. Are you recovering many or just a few colonies? How big is the insert? If you're only picking up these, it suggests a severe problem with your cloning. I assume your digest looks good. Now wanting to insult you... but I've done this myself: are you sure you are using the correct antibiotic? if both plasmids use a different selection, and you're inadvertently plating your ligations with the antibiotic needed for pRFP, then you'd get a result like you're describing. If that is ok, are you sure your ligation has worked? Do you have a positive control? Jose From wxfhome from gmail.com Sat Dec 15 20:20:51 2007 From: wxfhome from gmail.com (WANG XF) Date: Sat Dec 15 21:12:39 2007 Subject: About signal peptide Message-ID: <404daa2f0712151720t55075688j78b2ebf4cb593c93@mail.gmail.com> Hello, everyone! I have a question about protein secretion. I want to develop a construct as follows. *promoter----protein 1--protease site---signal peptide---protein 2* If the two proteins was expressed and cleaved at the protease site, protein 2 will be secreted into the supernatant? Thank you so much! LAO Wang From sudhee26 from gmail.com Mon Dec 17 10:23:18 2007 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Mon Dec 17 14:35:06 2007 Subject: IHC Related Problem Message-ID: Hello all, We are trying to do IHC on free floating 30 microns rat brain sections. Primary is antiGFAP (Mouse Monoclonal-1:1000) and secondary is antiMouse (Horse: 1:250), by ABC-DAB method. But we are getting reproducible nonspecific staining in SPECIFIC regions of the rat brain Our IHC method is 1. TBS / PBS Washing 2. Quenching for 10 min 3. TBS/ PBS washing 4. Blocking with 3%-5% Normal Horse Serum (also tried BSA) 5. Washing 6. Primary 7. Washing 8. Secondary 9. Washing 10. ABC 11. Washing 12. DAB We did a negative control by skipping the primary..but same results..the cells seem to be of pyramidal / immature neuron morphology. We also skipped both primary and secondary, going ahead with ABC-DAB but no staining (rules out endogenous biotin). We got convinced that only secondary can give nonspecific staining..we tried following methods-(Quenching-Blocking-Secondary) 1. increasing dilutions: from 1:250 to 1:1000 and then ABC-DAB..still we are getting the same nonspecific staining.. 2. Texas Red conjugated antiMouse (Horse) as secondary and got the similar pattern (no ABC method) 3. used antiRabbit(Goat) without primary..no staining (looks like problem is specific to antiMouse) 4. Reagents were changed, new vials (lots) were used, crucibles were changed..6-7 different brain sections were used..same result 5. This post-secondary antibody stain looks predominantly cytoplasmic, also present in axons 6. regions where staining is seen : somato sensory cortex, hippocampus, inferior temporal lobe, SVZ etc 7. Increasing blocking serum (Horse/Goat): 3 to 5%--same results 8. We have also tried NovaRed--same results. So, this antimouse antibody is binding to some proteins expressed in normal rat brain..?? in dire need of help. regards Deepti and Sudheendra NBRC, Gurgaon, India -- Think before agree Think before you nod but STOP thinking and You Are God From larisa_troitskaya from yahoo.com Mon Dec 17 19:39:28 2007 From: larisa_troitskaya from yahoo.com (Larisa) Date: Mon Dec 17 20:49:11 2007 Subject: removing bound SDS from protein samples Message-ID: <14375839.post@talk.nabble.com> Hi experts, I need very pure protein for FACE analysis. As last stage I'm going to use purification from SDS-PAGE: cut out single band from the gel and then extract protein. I'm afraid that SDS (still bound to my protein of interest after extraction) will interfere with PNGase F digestion (though reaction mixture contains SDS at final concentration 0.05%). Could you, please, suggest method to remove SDS from my protein sample after extraction SDS-protein complex from gel ??? By now I have found only info on GeBa kit, but it needs lots of buffer substitutions, and I could end up with unsufficient amount of protein. Thank you, Larisa -- View this message in context: http://www.nabble.com/removing-bound-SDS-from-protein-samples-tp14375839p14375839.html Sent from the Bio.net - Methods mailing list archive at Nabble.com. From sudhee26 from gmail.com Tue Dec 18 00:18:03 2007 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Tue Dec 18 13:24:11 2007 Subject: IHC Related Problem In-Reply-To: <710764ea0712171235g2fb9cd58y595e41f892b4426f@mail.gmail.com> References: <710764ea0712171235g2fb9cd58y595e41f892b4426f@mail.gmail.com> Message-ID: Hi POW, Susanne Thanks for the suggestions. 1. we have tried Alexa 594 conjugated sec 1:1000 2. Here we are not using primary at all..only sec gives this staining.. 3. we however wish to try this combination (primary 1:250, sec 1:2000) 4. we have done perfusion of rat prior to fixation with pbs.sections were directly put in DAB without quenching..but not significant staining was seen (?no or low endogenous peroxidase. also our staining was seen more in neuron like cells and not blood vessels-which indicates good perfusion) 5. we are not using formalin fixed tissues..we use paraformaldehyde to fix the animal tissue after pbs perfusion. 6. we have also tried different dilutions of biotinylated secondary. 7. we have also tried rabbit polyclonal antiGFAP..and in its -ve control stain (i.e without primary) no staining was seen as seen in antiMouse secondary earlier..that makes it specific to antiMouse. 8. the protein atlas is very nice..but regional pattern cant be seen..i guess they used punched tissue for staining. 9. we will send the pic if needed. still in dire need of help. Deepti and Sudheendra. On 12/18/07, Pow Joshi wrote: > > > > On 17/12/2007, Sudheendra Rao N R wrote: > > > > Hello all, > > We are trying to do IHC on free floating 30 microns rat brain sections. > > Primary is antiGFAP (Mouse Monoclonal-1:1000) and secondary is antiMouse > > (Horse: 1:250), by ABC-DAB method. > > But we are getting reproducible nonspecific staining in SPECIFIC regions > > of > > the rat brain > > > > Our IHC method is > > 1. TBS / PBS Washing > > 2. Quenching for 10 min > > 3. TBS/ PBS washing > > 4. Blocking with 3%-5% Normal Horse Serum (also tried BSA) > > 5. Washing > > 6. Primary > > 7. Washing > > 8. Secondary > > 9. Washing > > 10. ABC > > 11. Washing > > 12. DAB > > > > We did a negative control by skipping the primary..but same results..the > > cells seem to be of pyramidal / immature neuron morphology. > > We also skipped both primary and secondary, going ahead with ABC-DAB but > > no > > staining (rules out endogenous biotin). > > > > We got convinced that only secondary can give nonspecific staining..we > > tried > > following methods-(Quenching-Blocking-Secondary) > > 1. increasing dilutions: from 1:250 to 1:1000 and then > > ABC-DAB..still we are getting the same nonspecific staining.. > > 2. Texas Red conjugated antiMouse (Horse) as secondary > > and > > got the similar pattern (no ABC method) > > 3. used antiRabbit(Goat) without primary..no staining > > (looks like problem is specific to antiMouse) > > 4. Reagents were changed, new vials (lots) were used, > > crucibles were changed..6-7 different brain sections were used..same > > result > > 5. This post-secondary antibody stain looks > > predominantly > > cytoplasmic, also present in axons > > 6. regions where staining is seen : somato sensory > > cortex, > > hippocampus, inferior temporal lobe, SVZ etc > > 7. Increasing blocking serum (Horse/Goat): 3 to > > 5%--same > > results > > 8. We have also tried NovaRed--same results. > > > > So, this antimouse antibody is binding to some proteins expressed in > > normal > > rat brain..?? > > > > > I don't know too much about DAB staining ... however I have used the > secondary at 1:1000 dilution for alexa 488 or 594 conjugated with fairly > specific staining....perhaps the endogenous peroxidase was'nt properly > "quenched" ... check if the hydrogen peroxide is really good (If you have'nt > already done so). I would also suggest using the priomary at higher > concentration of 1:250 and lower secondary rather than the other way around > ..... ca'nt think of anything else for the moment. > > best, > Pow > > > in dire need of help. > > > > regards > > Deepti and Sudheendra > > NBRC, Gurgaon, India > > > > > > -- > > 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 > > > > -- Think before agree Think before you nod but STOP thinking and You Are God From sudhee26 from gmail.com Tue Dec 18 00:22:48 2007 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Tue Dec 18 13:24:17 2007 Subject: IHC Related Problem In-Reply-To: References: <710764ea0712171235g2fb9cd58y595e41f892b4426f@mail.gmail.com> Message-ID: Also.. we always prepare primary in blocking..so after blocking we give a mild wash with PBS...and then put primary..so that washing step is not to be taken seriously. Deepti and Sudheendra. On 12/18/07, Sudheendra Rao N R wrote: > > Hi POW, Susanne > > Thanks for the suggestions. > > 1. we have tried Alexa 594 conjugated sec 1:1000 > 2. Here we are not using primary at all..only sec gives this staining.. > 3. we however wish to try this combination (primary 1:250, sec 1:2000) > 4. we have done perfusion of rat prior to fixation with pbs.sections were > directly put in DAB without quenching..but not significant staining was seen > (?no or low endogenous peroxidase. also our staining was seen more in neuron > like cells and not blood vessels-which indicates good perfusion) > 5. we are not using formalin fixed tissues..we use paraformaldehyde to fix > the animal tissue after pbs perfusion. > 6. we have also tried different dilutions of biotinylated secondary. > 7. we have also tried rabbit polyclonal antiGFAP..and in its -ve control > stain (i.e without primary) no staining was seen as seen in antiMouse > secondary earlier..that makes it specific to antiMouse. > 8. the protein atlas is very nice..but regional pattern cant be seen..i > guess they used punched tissue for staining. > 9. we will send the pic if needed. > > still in dire need of help. > > Deepti and Sudheendra. > > > > On 12/18/07, Pow Joshi wrote: > > > > > > > > On 17/12/2007, Sudheendra Rao N R < sudhee26@gmail.com> wrote: > > > > > > Hello all, > > > We are trying to do IHC on free floating 30 microns rat brain > > > sections. > > > Primary is antiGFAP (Mouse Monoclonal-1:1000) and secondary is > > > antiMouse > > > (Horse: 1:250), by ABC-DAB method. > > > But we are getting reproducible nonspecific staining in SPECIFIC > > > regions of > > > the rat brain > > > > > > Our IHC method is > > > 1. TBS / PBS Washing > > > 2. Quenching for 10 min > > > 3. TBS/ PBS washing > > > 4. Blocking with 3%-5% Normal Horse Serum (also tried BSA) > > > 5. Washing > > > 6. Primary > > > 7. Washing > > > 8. Secondary > > > 9. Washing > > > 10. ABC > > > 11. Washing > > > 12. DAB > > > > > > We did a negative control by skipping the primary..but same > > > results..the > > > cells seem to be of pyramidal / immature neuron morphology. > > > We also skipped both primary and secondary, going ahead with ABC-DAB > > > but no > > > staining (rules out endogenous biotin). > > > > > > We got convinced that only secondary can give nonspecific staining..we > > > tried > > > following methods-(Quenching-Blocking-Secondary) > > > 1. increasing dilutions: from 1:250 to 1:1000 and > > > then > > > ABC-DAB..still we are getting the same nonspecific staining.. > > > 2. Texas Red conjugated antiMouse (Horse) as > > > secondary and > > > got the similar pattern (no ABC method) > > > 3. used antiRabbit(Goat) without primary..no staining > > > > > > (looks like problem is specific to antiMouse) > > > 4. Reagents were changed, new vials (lots) were used, > > > > > > crucibles were changed..6-7 different brain sections were used..same > > > result > > > 5. This post-secondary antibody stain looks > > > predominantly > > > cytoplasmic, also present in axons > > > 6. regions where staining is seen : somato sensory > > > cortex, > > > hippocampus, inferior temporal lobe, SVZ etc > > > 7. Increasing blocking serum (Horse/Goat): 3 to > > > 5%--same > > > results > > > 8. We have also tried NovaRed--same results. > > > > > > So, this antimouse antibody is binding to some proteins expressed in > > > normal > > > rat brain..?? > > > > > > > > > > I don't know too much about DAB staining ... however I have used the > > secondary at 1:1000 dilution for alexa 488 or 594 conjugated with fairly > > specific staining....perhaps the endogenous peroxidase was'nt properly > > "quenched" ... check if the hydrogen peroxide is really good (If you have'nt > > already done so). I would also suggest using the priomary at higher > > concentration of 1:250 and lower secondary rather than the other way around > > ..... ca'nt think of anything else for the moment. > > > > best, > > Pow > > > > > > in dire need of help. > > > > > > regards > > > Deepti and Sudheendra > > > NBRC, Gurgaon, India > > > > > > > > > -- > > > 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 > > > > > > > > > > -- > 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 valeriarix from libero.it Tue Dec 18 08:55:11 2007 From: valeriarix from libero.it (valeriarix@libero.it) Date: Tue Dec 18 13:24:22 2007 Subject: western blot Message-ID: <19851128.54361197986111154.JavaMail.nabble@isper.nabble.com> I forgot to put the DTT in western blot samples I can't have another go because I haven't other samples without DTT the western don't go or can I use it? thanks valeria From valeriarix from libero.it Tue Dec 18 09:01:44 2007 From: valeriarix from libero.it (valeria) Date: Tue Dec 18 13:25:32 2007 Subject: western blot Message-ID: <14397515.post@talk.nabble.com> I forgot to put the DTT in western blot samples i can't have another go the western blot is it destroy or can I use it? thanks valeria -- View this message in context: http://www.nabble.com/western-blot-tp14397515p14397515.html Sent from the Bio.net - Methods mailing list archive at Nabble.com. From pow.joshi from gmail.com Tue Dec 18 13:59:40 2007 From: pow.joshi from gmail.com (Pow Joshi) Date: Tue Dec 18 15:18:30 2007 Subject: western blot In-Reply-To: <14397515.post@talk.nabble.com> References: <14397515.post@talk.nabble.com> Message-ID: <710764ea0712181059v476b801cu483444b8fd63ad14@mail.gmail.com> On 18/12/2007, valeria wrote: > > > I forgot to put the DTT in western blot samples > i can't have another go > the western blot is it destroy or can I use it? > thanks > valeria you can still interpret the data depending on the locations of your -s-s- linkages in the protein. DTT or 2-ME are reducing agents that break th -s-s- bons to form -SH bonds. For eg: if you have antibody sample, the reduced Ab runs as two bands of ~55kDa and 25kDa while the non-reduced sample will run at ~150 kDa. Hope that helps Pow -- > View this message in context: > http://www.nabble.com/western-blot-tp14397515p14397515.html > Sent from the Bio.net - Methods mailing list archive at Nabble.com. > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From novalidaddress from nurfuerspam.de Tue Dec 18 16:50:35 2007 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Dec 18 17:30:44 2007 Subject: western blot References: Message-ID: <1479089b-0cc1-4051-aff6-a2dd19d1b1c7@d4g2000prg.googlegroups.com> Valeria, if you haven't loaded your samples yet on the gel, just add 5 or 10ul 2M DTT, it will work fine. If you already have run the gel, don't panic (too much), you just did not reduce your samples. They will run at different sizes maybe. You also might see some addional bands due to cross-linking and inter-/intramolecular -SS- bridges. Best, Wo From nobody from nospam.not Tue Dec 18 21:10:06 2007 From: nobody from nospam.not (Han) Date: Tue Dec 18 22:52:32 2007 Subject: western blot References: <1479089b-0cc1-4051-aff6-a2dd19d1b1c7@d4g2000prg.googlegroups.com> Message-ID: WS wrote in news:1479089b-0cc1-4051-aff6- a2dd19d1b1c7@d4g2000prg.googlegroups.com: > Valeria, > > if you haven't loaded your samples yet on the gel, just add 5 or 10ul > 2M DTT, it will work fine. If you already have run the gel, don't > panic (too much), you just did not reduce your samples. They will run > at different sizes maybe. You also might see some addional bands due > to cross-linking and inter-/intramolecular -SS- bridges. > > Best, > > Wo In addition, some antibodies don't recognize reduced proteins - they need a "native", folded protein configuration. -- Best regards Han email address is invalid From cintia.marques from ufjf.edu.br Wed Dec 19 07:39:08 2007 From: cintia.marques from ufjf.edu.br (=?iso-8859-1?Q?C=EDntia_Marques_Coelho?=) Date: Wed Dec 19 14:51:46 2007 Subject: cDNA RDA protocol Message-ID: <3740.200.131.61.61.1198067948.squirrel@correio.ufjf.edu.br> Dear Mike Hubank and David Schatz My name is C?ntia Coelho, I am a professor at the Federal University of Juiz de Fora, Brazil. I am just starting a new research line in my laboratoty to work with differentially expressed genes in prokaryote.Could you please send me a copy of the detailed RDA protocol? Thank you very much for the attention. Best regards, C?ntia --- PhD C?ntia Marques Coelho Federal University of Juiz de Fora Minas Gerais Brazil zip code: 36036-900 e-mail cintia.marques@ufjf.edu.br phone: 55 32 3229-3206 extension: 211 -- Profa. Adj. C?ntia Marques Coelho Universidade Federal de Juiz de Fora Departamento de Biologia Bairro Martelos 36036-900, Juiz de Fora, MG fone: (32)3229-3206 ramal:211 fax: (32)3229-3216 e-mail: cintia.marques@ufjf.edu.br From stewjw from gmail.com Thu Dec 20 05:28:21 2007 From: stewjw from gmail.com (StewJW) Date: Thu Dec 20 11:25:59 2007 Subject: IHC Related Problem References: <710764ea0712171235g2fb9cd58y595e41f892b4426f@mail.gmail.com> Message-ID: <6f4feebe-bade-4da7-b2e9-994040b2bb85@p69g2000hsa.googlegroups.com> Could it just be your primary antibody causing the problem? Zymed (Invitrogen) sells a primary mouse anti-GFAP antibody, catalogue 08-0021, which comes in a range of concentrations to work with your own development system or you can use one of Zymeds kits, which have a low background and very high sensitivity, The Histostain Plus Kit is a peroxidase DAB kit for mouse primary antibodies, catalogue 85-9143. These kits use a biotin labeled secondary antibody followed by a peroxidase labelled strepatavidin. The reagents come in dropper bottles and no reconstitution or pipetting is required, and they are designed specificially for IHC. Link for primary antibody product note: http://www.invitrogen.com/content/sfs/manuals/08-0021_Rev%201107.pdf From nick.theodorakis from gmail.com Thu Dec 20 10:36:30 2007 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Thu Dec 20 11:26:07 2007 Subject: IHC Related Problem References: Message-ID: <007020ca-76c0-4fc1-b5e9-bcbc33d435df@e25g2000prg.googlegroups.com> On Dec 17, 10:23 am, "Sudheendra Rao N R" wrote: > Hello all, > We are trying to do IHC on free floating 30 microns rat brain sections. > Primary is antiGFAP (Mouse Monoclonal-1:1000) and secondary is antiMouse > (Horse: 1:250), by ABC-DAB method. > But we are getting reproducible nonspecific staining in SPECIFIC regions of > the rat brain [...] > We did a negative control by skipping the primary..but same results..the > cells seem to be of pyramidal / immature neuron morphology. > We also skipped both primary and secondary, going ahead with ABC-DAB but no > staining (rules out endogenous biotin). Is your secondary (anti-mouse) antiserum guaranteed to be free of cross-reactivity to rat antibodies? Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From sudhee26 from gmail.com Thu Dec 20 23:47:37 2007 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Fri Dec 21 12:53:22 2007 Subject: IHC Related Problem In-Reply-To: <6f4feebe-bade-4da7-b2e9-994040b2bb85@p69g2000hsa.googlegroups.com> References: <710764ea0712171235g2fb9cd58y595e41f892b4426f@mail.gmail.com> <6f4feebe-bade-4da7-b2e9-994040b2bb85@p69g2000hsa.googlegroups.com> Message-ID: Hi StewJW, Nick thanks for the suggestions. Actually as we told, we have also tried other company anti-GFAP, anti-S100b primary. (we used anti-s100b because it can selectively stain glial cells..not like GFAP..immature neurons might express GFAP..so with the former we expect only glial cells..but in negative control we are seeing neurons!!!--pyramidal in cortex and hippocampus) when we use primary we dont get this pattern..its only when we use secondary alone. As Nick was pointing out it cant be said that our secondary is free from interacting with rat antibodies. We use vector biotinylated secondaries and we have tried a different lot to end up with same results. We can however try with vector elite ABC kit.. PS: we have use Fluorescein Conjugated( Rat Adsorbed )antiMouse..but same results in Negative Control!! that rules out antirat mouse antibodies in secondary. we would say..its not the rat antibodies the secondary is interacting with.it some rat protein which has homology to mouse protein..hence antimouse is binding to it..not quite sure.. We tried 10% blocking horse serum, Primary 1:1000 and secondary 1:2000 dilution..but same results in the negative control. Can any one tell me how does a secondary antibody interact..as in through Fc portion?? How do antiMouse, antiRabbit, antiGoat secondaries differ?? Sudheendra and Deepti. On 12/20/07, StewJW wrote: > > Could it just be your primary antibody causing the problem? Zymed > (Invitrogen) sells a primary mouse anti-GFAP antibody, catalogue > 08-0021, which comes in a range of concentrations to work with your > own development system or you can use one of Zymeds kits, which have a > low background and very high sensitivity, The Histostain Plus Kit is a > peroxidase DAB kit for mouse primary antibodies, catalogue 85-9143. > These kits use a biotin labeled secondary antibody followed by a > peroxidase labelled strepatavidin. The reagents come in dropper > bottles and no reconstitution or pipetting is required, and they are > designed specificially for IHC. > > Link for primary antibody product note: > http://www.invitrogen.com/content/sfs/manuals/08-0021_Rev%201107.pdf > _______________________________________________ > 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 liangxin28 from gmail.com Thu Dec 27 22:55:35 2007 From: liangxin28 from gmail.com (Tay liang xin) Date: Fri Dec 28 14:02:10 2007 Subject: removing bound SDS from protein samples (Larisa) Message-ID: <42b3ff70712271955h47116dbfja7d294d098c0691b@mail.gmail.com> Dear Larisa, Maybe you can refer to this journal doi: 10.1016/j.jmb.2007.11.026 to solve your problem. "Refolding SDS-Denatured Proteins by the Addition of Amphipathic Cosolvents" Regards Liangxin INFORMM, USM Malaysia From sarita81 from gmail.com Fri Dec 28 00:46:26 2007 From: sarita81 from gmail.com (sarita mallik) Date: Fri Dec 28 14:02:23 2007 Subject: transport s bacterial strain Message-ID: Dear All, I 'm a research scholar in India. i have to go to Germany for a 6 month reserch fellowship. for this i need to carry my bacterual strain (non pathogenic) along with me. can anybody suggest the guidelines for export of biological material . thanks Sarita From sarita81 from gmail.com Sat Dec 29 00:25:01 2007 From: sarita81 from gmail.com (sarita mallik) Date: Sat Dec 29 12:48:41 2007 Subject: transport s bacterial strain In-Reply-To: References: Message-ID: Dear all Thanks for the suggestions. Do we need an authority letter or some sort of consent letter from university at india and the institute at geermany. Thanks sarita On Dec 29, 2007 2:20 AM, DK wrote: > "sarita mallik" wrote: > >Dear All, > >I 'm a research scholar in India. i have to go to Germany for a 6 month > >reserch fellowship. for this i need to carry my bacterual strain (non > >pathogenic) along with me. can anybody suggest the guidelines for export > of > >biological material . > > Regulations in India, Germany and airlines aside, this can be > as trivial as taking a diluted LB culture into screw cap eppendord > and putting it in the pocket. Or you can bring a streaked plate with > you. Or a lyophilized sample (add just about any sugar to >1M > and lyophilize). > > But, because of the regulations crap, I'd probably send the culture > by Fedex to a colleague in Germany. > > DK > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From sudhee26 from gmail.com Mon Dec 31 00:27:46 2007 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Mon Dec 31 13:12:22 2007 Subject: transport s bacterial strain In-Reply-To: References: Message-ID: Hi, Go through the following..might help you. http://www.uiowa.edu/~hpo/biosafety/sisfaq.pdf http://www.uiowa.edu/~hpo/biosafety/SISintro.pdf http://www.uiowa.edu/~hpo/biosafety/freqshipmat.pdf http://www.fedex.com/in/services/dangerousgoods.html Sudheendra. On Dec 29, 2007 10:55 AM, sarita mallik wrote: > Dear all > Thanks for the suggestions. Do we need an authority letter or some sort of > consent letter from university at india and the institute at geermany. > > Thanks > > sarita > > > On Dec 29, 2007 2:20 AM, DK wrote: > > > "sarita mallik" wrote: > > >Dear All, > > >I 'm a research scholar in India. i have to go to Germany for a 6 month > > >reserch fellowship. for this i need to carry my bacterual strain (non > > >pathogenic) along with me. can anybody suggest the guidelines for export > > of > > >biological material . > > > > Regulations in India, Germany and airlines aside, this can be > > as trivial as taking a diluted LB culture into screw cap eppendord > > and putting it in the pocket. Or you can bring a streaked plate with > > you. Or a lyophilized sample (add just about any sugar to >1M > > and lyophilize). > > > > But, because of the regulations crap, I'd probably send the culture > > by Fedex to a colleague in Germany. > > > > DK > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > _______________________________________________ > 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 virashkgupta from gmail.com Mon Dec 31 03:09:19 2007 From: virashkgupta from gmail.com (Virash Gupta) Date: Mon Dec 31 13:12:28 2007 Subject: Methods Digest, Vol 31, Issue 17- Re: transport s bacterial strain Message-ID: This is common requirement and practice amongst scientists who move amongst different countries. I am using following methods. 1. Dispense LB-agar in 1.5 ml sterile microcentrifuge tubes, let them solidify in verticle position to make stabs. You can add antibiotic if you know your culture has resistance for it in media before dispensing (this will keep contaminants in check). Pick small amount of overnight grown culture with a sterile loop and insert the needle vertically into solidified stab. Close the tube and carry as amany as you can in small space. Ypu can easily retrieve the culture for upto 15 days if carried at RT or upto 3 month if stored at 4 degree C, after picking small growth from the top of stab from inside thestab by streaking on the plate. It will not fail. LB allows growth of most organisms- or you can use optimal medium. 2. Alternately mix one volume of good liquid culture with half volume of 50% sterile growth. close the tube and carry. Shuold not be difficult/ all the best Dr V K Gupta Professor of Microbiology and Mol Biology PAU, Ludhiana virashkgupta@gmail.com On Dec 29, 2007 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. Re: removing bound SDS from protein samples (Larisa) > (Tay liang xin) > 2. transport s bacterial strain (sarita mallik) > 3. Re: transport s bacterial strain (DK) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Fri, 28 Dec 2007 11:55:35 +0800 > From: "Tay liang xin" > Subject: Re: removing bound SDS from protein samples (Larisa) > To: methods@oat.bio.indiana.edu > Message-ID: > <42b3ff70712271955h47116dbfja7d294d098c0691b@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Dear Larisa, > > Maybe you can refer to this journal doi: 10.1016/j.jmb.2007.11.026 to > solve > your problem. > "Refolding SDS-Denatured Proteins by the Addition of Amphipathic > Cosolvents" > > Regards > Liangxin > INFORMM, USM > Malaysia > > > ------------------------------ > > Message: 2 > Date: Fri, 28 Dec 2007 11:16:26 +0530 > From: "sarita mallik" > Subject: transport s bacterial strain > To: methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Dear All, > I 'm a research scholar in India. i have to go to Germany for a 6 month > reserch fellowship. for this i need to carry my bacterual strain (non > pathogenic) along with me. can anybody suggest the guidelines for export > of > biological material . > > thanks > > Sarita > > > ------------------------------ > > Message: 3 > Date: Fri, 28 Dec 2007 20:50:45 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: transport s bacterial strain > To: methods@net.bio.net > Message-ID: > > "sarita mallik" wrote: > >Dear All, > >I 'm a research scholar in India. i have to go to Germany for a 6 month > >reserch fellowship. for this i need to carry my bacterual strain (non > >pathogenic) along with me. can anybody suggest the guidelines for export > of > >biological material . > > Regulations in India, Germany and airlines aside, this can be > as trivial as taking a diluted LB culture into screw cap eppendord > and putting it in the pocket. Or you can bring a streaked plate with > you. Or a lyophilized sample (add just about any sugar to >1M > and lyophilize). > > But, because of the regulations crap, I'd probably send the culture > by Fedex to a colleague in Germany. > > DK > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 31, Issue 17 > *************************************** > -- 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 neal.melvin from gmail.com Mon Dec 31 22:10:15 2007 From: neal.melvin from gmail.com (neal.melvin@gmail.com) Date: Tue Jan 1 11:58:59 2008 Subject: Confirming successful cDNA synthesis Message-ID: <6349829d-268f-45f8-b8aa-3e6a2ea9031f@e6g2000prf.googlegroups.com> Hello, I am just starting reverse transcription-based PCR in a new lab (it's the first time that PCR has been attempted here). I have been using the Protoscript II kit from NEB, but have not seen any bands on my gel in the end, even using the control rat liver RNA and GAPDH PCR primers and conditions included in the kit. Incidentally, I have primed the RT using the supplied anchored oligo dTs. I would like to know whether the RT step or the PCR is the problem. I can't get my hands on any other general PCR primers to other genes at the moment, which is what I would use to confirm that the RT went well. I have confirmed that the control total RNA included in the kit is good, as assayed by running a denaturing RNA gel: both the 28s and 18s bands were very strong. >From what I've read, the typical way to confirm a successful RT is to include radiolabelled nucleotides during the RT step, and then analyse the products on a gel afterwards. Since I'd rather not resort to radioactivity-based experiments, is there any other way that I can use to confirm that I actually have cDNA? I did try to run a large amount of the RT reaction on a gel itself to look for signs of cDNAs of various lengths, but saw nothing. I suppose one reason that I saw nothing is that the cDNA may have been too dilute... can I use standard ethanol precipitation to precipitate and concentrate my supposed cDNA sample? From skatz from rsu.edu Mon Dec 31 17:22:02 2007 From: skatz from rsu.edu (Sue Katz) Date: Tue Jan 1 11:59:05 2008 Subject: Looking for copy of PFGE apparatus manual Message-ID: <26AF74286FCA14429EA92AD72A3276220605B3DA@rsufsmail.rsu.edu> Hello I've just gotten a used BRL (Life Sciences) Hex-a-field apparatus. If anyone has a copy of the manual that they can send me, I would appreciate it. Thank you Sue Katz, Rogers State University, Claremore, OK