Help with RT-PCR of 1.4Kb fragment - (Oct/13/2008 )
I am trying to amplify the promoter region of my gene. I have designed primers which amplify the whole promoter region, as well as the 1st half and 2nd half of the promoter. I have had success with amplifying the two halves of the promoter but have had no success when trying to amplify the whole promoter.
The length of the full promoter fragment is 1.4kB and I am using GoTaq from Promega. The length of the primers is 20-mer with 50% GC content. The Tm for the forward primer is 59.4 and the reverse is 57.3. Tm have been confirmed, both by hand and with online programs. The program I am using is as follows:
> 95; 5mins
> 95; 15secs
> 50; 15secs
> 72; 2.8mins
> 72; 10mins
> 30 cycles
I have increased with amounts of dNTPs, enzyme and MgCl2, but with no success, other than primer-dimers. The reverse primer used to amplify the 2nd half of the promoter is also used for the full-lenght promoter and while it works with this region, it doesn't with the full promoter.
I would greatly appreciate any advice (other than crossing-fingers and praying) as I am now developing a bald spot on the back of my head from pulling my hair.
All the best,
P.S. Tried Pfu Polymerase with the same conditions described, but got a smear.
I think you are not using enough time for annealing. and with those tm i suggest this
72 1min 30s
check the sequence of the primers!!
Go taq could be that with out problem. If not try with taq platinum
Check whether promoter region u are trying to amplyfy is GC rich or AT rich.
And also u can try with different proof reading taq's available at your lab. I had a bad experience with my promoter isolation and atlast i succeeded when i standardized using ID proof.
Hope this helps.