PCR crash: Feedback!
PANIAGUA SOLIS JORGE FERNANDO
jpaniagf at CONDOR.DGSCA.UNAM.MX
Wed Nov 16 12:02:48 EST 1994
From 0 to 3 ug DNA in 2 days.
One month ago I posted a unexplained failure of a PCR reaction. I hope
this feedback may be helpful for someone(like me).
1)The main reason of the failure was the enzyme. Our BRL batch went bad.
How can one be sure of the activity if only have one set of primers?
2) The Mg concentration didnt modify drastically the yield of the band,
but how deeply it affects the fidelity of the enzyme?
3) The first time I got bands was using a two-step PCR. Amplify, take an
alicuot and reamplify in a fresh reaction. Many thanks to Leon
4) I wonder why the crude(really crude) primers work much better that the
gel purified ones.
5) I am using the robocycler 40 from Stratagene; I never got bands for
the "control" reaction , it was done in a Perkin Elmer machine. Does the
ramping machines give widers tolerance to the parameters of the reaction?
6) Many thanks to the enthusiastic participation of everybody.
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