genomic DNA PCR
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
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