gDNA PCR - for promoter amplification (Jun/13/2007 )

Hi,
I've been trying to amplify a 1800bp promoter region from a human gene. I extracted DNA from HeLa and Mia Paca cells.
I performed a 40 cycles program with extension temperature 55ºC for 2.30 minutes.....
I used Taq and all the standard things added..
I got a band for the control.......amplification of a 300bp actin gene for both cell lines but no amplification of the promoter...
What can I do?

If the problem is that the fragment is too long for the Taq, should I used a mixture of enzymes? which ones? Pfu and Taq works?

I read in some cases DMSO is added, is it worth trying? I checked the GC content but it has more or less the same amount of bases, so is DMSO useful?

looking for help!!

biotech

-biotech!-

QUOTE (biotech! @ Jun 13 2007, 04:22 PM)

If the problem is that the fragment is too long for the Taq, should I used a mixture of enzymes? which ones? Pfu and Taq works?

I read in some cases DMSO is added, is it worth trying? I checked the GC content but it has more or less the same amount of bases, so is DMSO useful?

looking for help!!

biotech

hi biotech,
Extension temperature should be at 72ºC. Generally, a 1.8kb amplicon is not too long for Taq. You could try to change annealing temperture. I usually set it at Tm+5ºC.

-WHR-