TC1 deletion PCR: artifacts

William R. Morgan wmorgan at ACS.WOOSTER.EDU
Tue Aug 16 15:23:39 EST 1994


Hello worm breeders,

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?

Some additional details:

Worm lysates from both N2 and YK8 (mut-2; kin-16::Tc1) animals are prepared
essentially as described by Burglin et al. [WBG 13(1):28].

25 ul PCR is performed with 1 ul lysate, 3.5 mM MgCl2 and 50 pmol each
primer,etc.  The primers are a 22mer (45%GC, Tm=58.5o) and a 21mer (52%GC,
Tm=61.7) normally separated by 2.9kb (4.5 kb in Tc1 strain).  [MgCl2] from
2-4.5 mM were tested; all give 1 kb artifact.

After a "hot start", I add 1.5 U Taq Pol + 0.25 U SSB protein (USB's
Bind-Aid), then do 35 cycles (30s @ 94o,1' @ 55o, 2' @ 72o).

Thanks for any suggestions.  You can send your replies directly to me and
I'll post a summary in a week or so.

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 at acs.wooster.edu




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