PCR crash

Wed Oct 26 16:53:33 EST 1994

Hello, colleages.
I am stuck with an amplification:
I need to amplify a 1 kb band from E. coli genome. I designed the 22 and 
26 b primers long with 3 mismatches very close to the 5' end. Their 
theoretical Tm is 57-60 C. I am using phenol-chloroform purified template 
and tried between 15 ng to 0.15 pg, diluted with sterile water.I have moved 
concentration of primers, dNTP's and temperature down to 35 degrees. I'm 
using the robocycle machine and the region to be apmlified is close to a 
promotor from the 5' and around a taatga region. Someone in the lab made 
it work but with 20 mM of MgCl2 in the reaction. Before I try it, I would 
be very glad to hear any sugestions.  Thank you to all of you for your 
help.     Jorge Paniagua  jpaniagf at condor.dgsca.unam.mx

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