Yet more PCR problems
leij at sage.cc.purdue.edu
Tue Feb 7 22:12:37 EST 1995
smori at nmsu.edu (Shahram Mori) wrote:
> LMS (Tel 0635 578411 X2598) (lee.smith at afrc.ac.uk) wrote:
> : UNTIL: just before christmas, I ran a titration of target,
> : and all I got were smears. I repeated it (twice), I got smears.
> : I changed all my reagents, smears. I changed my PCR machine, smears.
> : I went back to the beginning, and used my upstream primer with the
> : other two downstream primers- worked perfectly. A Ha I cried, my
> : primer has gone funny. I ordered more primers. They arrived.
> : I used them. Smears again (and sometimes, absolutely nothing).
> Could it be that your DNA has fragmented? It might be that there is a
> contaminating piece of DNA [ a fragment of the original] where the primer
> binds to and that gives you smear. I would recommend running your DNA on
> agarose to see if there are any other contaminating DNA's there. This is just
> an idea, probably a long shot.
I had a similar situation as encountered by Lee before. It turned out that the
problem was the change of Taq from company A to company B. Since I decided not
to reoptimize the PCR condition just for the new Taq, I went back to the
orginal Taq and my day was back to normal. I am not sure if this is Lee's
Postdoc, Horticulture Dept.
Speaking for myself..........
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