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

Duncan

 
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