Q: PCR not working

Duncan Clark Duncan at genesys.demon.co.uk
Tue May 2 07:16:40 EST 1995

In article: <3o0kbn$4vr at hydra.acs.ttu.edu>  Z3C20 at ttacs3.ttu.edu (Yi, Xiaoming) writes:
> HI, netters,
> I am using pfu enzyme. Recently, I had two 25mers made, but the PCR stopped
> working. I tried taq, which worked. I called Stratagene technical assistant, I
> was told that sodium inhibits the enzyme. I did a little calculation, I could
> not have that much sodium even if the primers I ordered were all sodium
> chloride. I can't figure out what is wrong. I made these two 25mers which are
> 100% complementary to the sites, no sequence with 70% homology found in the
> plasmid. Any help appreciated.

In my experience the proof-reading enzymes require a lot more optimisation of
Mg and Tm than normal Taq. A hot start may also be best. The 3'5' exo of these
enzymes has a nasty habit of chewing thge primers back to roughly 15 mers 
which no longer prime at the original Tm.

Good luck


My mind's made up. Don't confuse me with the facts!

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