Promoter cloning... - Problems (Jun/27/2008 )

hi guys, i am having some problems with my PCRs, i am trying to clone 4 promotors from human gene, with different concentrations of DMSO, 0%, 4% and 8%, however i have added a restriction enzyme site to my primers, and i tried severall Tms, starting at 55, 58 and 60, and it seems to NOT HAPPEN, i have changed 2 sets of primers already, and tried with different concentrations of MgCl2, but nothing seems to work, can anyone please help.

Thanks

Berty.

-Berti-

QUOTE (Berti @ Jun 27 2008, 06:44 AM)

1. chekc DNA quality

2. Make sure you give enough amplification time

3. See if the BAC clone is available for that genomic region, get it and use it as a template. Sequence check.

4. Try Nested PCR. If you find some better primers for a slightly bigger fragment, amplify that first (without RE tag), and use that template for your specific PCR.

5. Change your PCR Kit.

-cellcounter-

If you have GC-rich target, which it sounds like you do, try some betaine.

-killerkoz17-

thanks you for your answers.
if i get any results i will post them here :-).

-Berti-