genomic DNA PCR

clarosa clarosa at biocomp.unl.edu
Wed Oct 4 17:02:39 EST 2000


General protocol for PCR
Each person uses a slightly different ratio of reagents.

For DNA amplicons up to 500 bp, use 25 uL reaction volumes as follows:

On ice add to thin-walled pcr tubes

     0.125 uL Taq polymerase (1.25 U, Gibco)
     2.5 uL of 10 x PCR buffer minus Mg (1x)
     0.75 uL of 50 mM MgCl2  (1.5 mM)
     x uL template DNA (25-50 ng genomic dna)
     0.5 uL of 10 mM dNTP mix (0.2 mM)
     1.25 uL 10 uM primer mix (0.5 uM)
     water to 25 uL

If DNA amplicon is >500 bp, scale-up the reactions for 50 or 100 uL total
volume.  Program thermocycler appropriately and perform PCR reactions. 100
ng genomic dna is roughly right , depending on complexity, quality etc.


Robert Mihalek wrote:

> Can someone forward me a general protocol for doing PCR using genomic
> DNA as the template?  I seem to recall needing about 100 ng of DNA, but
> I forget stuff like  how much Taq to use, concentration of dNTP's, and
> concentration of primers.
>
> Many thanks!
>
> _______________________
> Bob Mihalek
> Mass. General Hospital
> Dept. of Anesthesia
> _______________________
> Views, comments, etc. are not representative of MGH or its affiliates






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