PCR from genomic dna

[Dr. Klaus Eimert]

Henri Spronk wrote:
> 
> The amount of genomic DNA we use in a typical PCR is normally about 300 ng.
> We find out that addition of 10 % DMSO to the PCR mixture improves the
> amplification of our fragments. Without DMSO the amplification failed a
> couple of times. Since DMSO helps to denature the genomic DNA, we always ad
> it to the PCR mixture now.
> 
> Henri Spronk
> Department of Biochemistry
> Mastricht University
> The Netherlands
> 

Hi,

300 ng sounds quiet a lot to me, what volume are we talking about? We
usually have no problem to amplify single copy genes with specific
primers from genomic plant DNA using 1 ng/µl DNA (e.g. 25 ng in 25 µl
total rxn volume). Once we accidently used undiluted DNA (endend up with
about 150 ng/µl rxn)and didn't get any distinct band - just smear. In
our hands DMSO usually is not necessary, unless we have some really
GC-rich stretches to amplify (then we use 5-10%).
Hope that helps,

Klaus 


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Dr. Klaus Eimert
Dept. of Botany
State Research Institute Geisenheim	Phone:    + 49 6722 502 469
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D-65366 Geisenheim			E-Mail:   eimert at web.de
Germany					URL: www.mnd.fh-wiesbaden.de/fag/bio/bo/ebostart.html
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