PCR crash

Emma Macfarlane emma at HERA.MED.UTORONTO.CA
Mon Oct 31 13:40:49 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
Hi Jorge
Have you tried increasing the amount of your template DNA?  In PCR 
reactions I have done using bacterial genomic DNA, I used 500ng of 
template (and 10mM MgCl2).  This may sound like overkill but it worked 
beautifully every time. 
Hope this is of some help

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