4 replies to this topic

[maza]

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.

[SVTX]

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

[thineric]

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...D=7&SubCatID=50

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

e-

[maza]

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

M