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.
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.
If you have GC-rich target, which it sounds like you do, try some betaine.
thanks you for your answers.
if i get any results i will post them here :-).