jeff at COMPBIO2.MED.WAYNE.EDU
Fri Jan 20 12:18:08 EST 1995
So heres my problem. (Sorry if my explitive in the subject heading
offended anyone). There was a grad student here in my lab who left last Fall.
He did about a million PCRs. When I joined here, having never performed PCR
before in my short, chemistry major little life, I worked my way through the
learning curve, sacrificed all the apropriate cute little animals to the PCR
gods, and pretty much got everything to work. Except for two primer sets. One
of them, primers for human actin, apear to have wroked every time Jim (the
grad student) used them. He used them at at least two different Ta's (60 and
63) and with many different buffers. They work fine.
So, I come along, and they DO NOT WORK. I have tried 50, 55, 57, 60,
63, 65 and 68 C; 40, 60 and 120 seconds annealing time; 1, 2 and 5 min extention times (1.2 kb product); buffer pH's from 8.5 to 10; Mg conc from 0.5 mM to 3.5
mM (final conc); AmSulf conc from 5 mM to 20 mM, 5 and 10 % glycerol; 5 and 10 %
DMSO; perfect match; 5 % gelatin, 50, 100, 200 and 500 ng of primer/rxn; 2.4,
24, 240 and 2400 ng genomic DNA (he used ~200); 100, 250, 500 uM and 1, 2.5, 5
and 10 mM final nTP conc; Tfl, Tth, taq and vent polymerases; hot start and no
hot start; two and three cycle amplifications; blah blah blah...
I used both old and new tubes of reagents, some from Jim's box, in case I was
making them wrong, and some new ones, in case his had gone bad. I have used up
an incredible amount of polymerase. The primers were run through Oligo a few
times, and they don't form any secondary structure, etc., and besides, they
worked for him. I believe I've tried every single well in my thermocycler. I
know my reagents aren't bad, because they work for every other primer set I
use. I have even tried growing a big cheesy mustache, like Jim, to try to fool
the PCR gods into thinking I'm him so the PCR will work. Well, I've narrowed
it down to two possibilities. I use my own genomic DNA, Jim used his. Either
A) I do not have an actin gene (unlikely, because I am alive), or
B) the primers are shot.
My boss tells me primers can't go bad. "What can happen to them?" he said.
Now as a chemist, I know the phosphodiester bond is relatively stable, but
something is definately wrong here. Remember, this is happening with two sets
of primers, so I doubt that someone just accidently spilled phenol in four tubes
of primer. These primers have always been taken care of, never left sitting
out over night, etc, but I am sure that somehow they are shot. It can't be
anything else, can it?
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