PCR crash: Feedback!

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 
Garcia-Martinez, UTSMC.
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.

More information about the Methods mailing list