From owner-celegans@net.bio.net Wed Sep 07 23:00:00 1994 Path: biosci!ACS.WOOSTER.EDU!wmorgan From: wmorgan@ACS.WOOSTER.EDU (William R. Morgan) Newsgroups: bionet.celegans Subject: SUMMARY: TC1 deletion PCR artifacts Date: 8 Sep 1994 14:05:32 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 128 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HGVL2I1Q428WYHFS@acs.wooster.edu> NNTP-Posting-Host: net.bio.net Earlier I wrote: I'm conducting a screen for Tc1-mediated deletions starting with a Tc1 insertion allele of kin-16 and I'm getting a major artifactual product of about 1 kb. Thomas Burglin and David Greenstein state that they see similar "non-specific bands that are also present in all reactions, but you should look for bands that occur only in one of the samples" (Burglin and Greenstein, "Tc1 Deletion PCR Tips" available by Gopher at weeds.mgh.harvard.edu). However, I'm concerned that the amount of the artifactual product (typically 25-75 ng/ 10 ul) may be out-competing the desired, rare deletion products. Should I worry about this PCR artifact or should I merrily continue my screen? Thomas Burglin and David Greenstein suggested making new primers. So I went back to my freezer, tried out what I had in all possible combinations, and found one that yielded essentially no artifactual bands. Anyway, shortly thereafter Yoshiki Andachi sent me the kin-16/kin-15 deletions I wanted which he had isolated from some practice runs he was doing. He had used a different set of primers which gave him no problems. Ron Plasterk and David Greenstein also recommended nested PCR using a second set of primers. Many thanks to those who replied: Thomas Burglin David Greenstein Michel Labouesse Jeffrey Yuan Ron Kagan David Eisenmann Ronald Plasterk Below, I've included a couple of letters about experiences with the TC1 excision screen that I thought people might find useful. ------------------------------------------------------------------------------ Hi, in response to your call for technical tips regarding the search for Tc1 excision, I can tell you what has been our experience with an Tc1 excision from the gene lin-26. 1) We had several background bands under 600 nt across all lanes, one of which @ 550 nt was quite strong. We did not worry about them and nonetheless got two excision events out of 125 initial pools screened (1 subsequently lost). 2) We basically followed Thomas Burglin's protocol: 30 sec @ 94~C, 60 sec @ 62~C, 2 min @ 72~C; no hot start; oligos were 23-mers roughly 50% G-C rich and naturally 2.3 kb apart. Therefore our conditions are not too different from yours except that we hybridized at a higher temp due to a higher GC content. 3) In the two lanes where a candidate excision had occured, the excision band was really obvious. In retrospect, it could not be confused with other artefactual bands. We nonetheless systematically checked the gels resulting from the first screenings by southern blotting with an internal oligo probe. 4) Given that the mut strains are sick, when you'll reach the final subcloning steps, ALWAYS try to sib-select from MORE THAN ONE positive pool. We almost lost our first positive deletion while going from plates started with 5 animals to plates started with 1 animal, we tried to sib-select from a single five- mother positive plate. 5) Never give up. Good luck. Michel Michel Labouesse U.184 - INSERM/ LGME - CNRS Faculte de Medecine 11 Rue Humann 67085 Strasbourg Cedex, France Tel (33) 88 37 12 55 Ext. 492 Fax (33) 88 37 01 48 ------------------------------------------------------------------------------- Hi Bill, I saw your note on bionet.celegans. I am just gearing up to screen for a deletion in my gene (hlh3). I have occasionally noticed the 1kb artifactual band though for the most part I have not had such a problem. The only time I have consistantly seen the 1kb band was doing a secondary screen of a TC1 library on another gene that I am interested in (hlh-4). The artifactual band appeared in at all dilutions in several samples. My conditions are similar to yours, though my MgCl2 concentration is 1.5mM and my annealling temperature is quite high (60C for 1 minute). What sort of primers are you using? If you are willing, I will be happy to test your primers on my worms to see if I also get the same 1kb band. Speak to you soon! Yours, Jeffrey Yuan ************************************************************************** * Jeffrey Yuan * * Department of Molecular Biology * * Lewis Thomas Laboratory * * Princeton University FAX: 609-258-3980 * * Washington Road Phone: 609-258-5939 * * Princeton, NJ 08544 email: yuan@phoenix.princeton.edu * ************************************************************************** Thanks again to all who replied and please pardon the delay in posting this summary. Bill Morgan William R. Morgan Assistant Professor The College of Wooster Department of Biology 931 College St. Wooster, OH 44691 Phone: 216-263-2026 FAX: 216-263-2378 E-mail: wmorgan@acs.wooster.edu .