PCR from genomic DNA
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Thu Sep 30 03:15:57 EST 1999
In article <email@example.com>, Regina Shaw
<regina at BIOTECH.UFL.EDU> writes
>Does anyone have suggestions as to what additives and/or polymerases worked
>best in their experience? Redesigning primers, optimizing Mg2+, and adding
>TMAC (even though the sequence is not particularly GC-rich) has helped
>alot, but the yields are still inconsistent - most templates amplify, but
>some don't. It's not concentration-dependent.
Assuming you have a target that has no high G/C (as you say it hasn't)
or any weird secondary structure then straight Taq should do fine for
your target size. Standard 1.25u/50ul PCR with 1.5mM Mg and appropriate
buffer should work. I assume the primers are checked by suitable primer
design software. What do you actually get on your failed wells, no
product at all or dimers or multiple products or what?
Can you be sure that the it is not the quality of your template that is
the problem. Could you spike your master mix with say 0.1-10pg of lambda
DNA and a 500bp lambda target primer pair. It will act as an internal
control target. If you do not get consistent amplification of the lambda
across all wells when your xsomal target and primers are present then
either an inhibitor is present in your sample or maybe your machine
block is out of spec. You should be able to design a lambda primer pair
that fit your existing annealing temp and won't interfere with your
xsomal primer pair etc.
As for alternative additives/polymerases to try. Additives include
betaine to 1M, DMSO up to 10%, polymerase mixes i.e. Thermus pol plus
proof-reading pol. Etc.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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