Poor PCR yield - (Aug/30/2004 )

Hey,

I have been having problems with the yield of my 2kb promoter PCR. I optimized a hot start method using ABgenes therostart Taq on test DNA but when I try to amplify my samples a few work (but at different intensities) and some just don't amplify at all. They were plated out by another group and are obviously all at different concentrations and I only have a limited supply. I have tried adding DMSO and varying the anealing temp but this just produces smaller unspecific products. Is there anything I can do to improve the yield? Any suggestions would be greatly appreciated.

My cycling conditions are:

95 - 15mins
95 - 30sec
67.7 - 30sec 45 cycles!!!
72 - 3min
72 - 7min

Thanks.

-maza-

Hi,

Try
95C-2min;
{94C-10s;52C (or your specific annealing temp)-1min;68C-2min}- 35cycles
68C-10 min

If you are using it for cloning, use high-fidelity Taq such as Pfu.

Good luck
SV

-SVTX-

Since you are amplifying a promoter region which is usually GC rich, GC rich seqeuences are nortoriously difficult to amplify. There are some measures you can take to get around this problem. You may want to read this paper

http://www1.qiagen.com/Literature/qiagenne...ng/default.aspx

-twister-

hi-
i had the same issue just a couple of months ago and i changed almost every single variable trying to get results, including the ones you mentioned...i eventually gave up and ordered a product called failsafe PCR and it worked wonders... you should check it out...it is surprisingly inexpensive http://www.epicentre.com/item.asp?ID=294&CatID=7&SubCatID=50

oh and i also got a higher yield after lowering my primer concentration.

e-

-thineric-

Thank you for all your suggestions - I shall keep trying!!!

M

-maza-