PCR problem (great different of Tm in primers) - (Jan/06/2009 )

I am going to clone a gene by amplifying the cDNA by PCR with designed primer.

However, there is a great different between the Tm for the forward primer and the reverse primer, and the product size should be 1503 bp and 1351 bp respectively.

1. Forward primer: 85.02'C
Reverse primer: 68.13'C

2. Forward primer: 78.66'C
Reverse primer: 68.13'C

What is the suitable annealing temperture should i take for my PCR?

The following is my condition used before:

94'C 5mins

94'C 30s
55'C 30s 25 cycles
72'C 2mins

72'C 7mins
4'C hold

Also, i have try 60'C annealing temperture.
Both pairs of primer and temperture show no band.

Hi

I would suggest that you reengineer one of the primers to get the Tms close to each other. If you still wana try this, you can try annealing at 65 or try another enzyme. When i setup a PCR in hurry, I just choose 4 degrees less than the lower Tm, It works in most of the cases.

TC

You should try a product called PCRboost. I get it from Biomatrica. I have fallen in love with it. You basically use it instead of water and it increases the yeild and performace of the PCR reaction. Try it, you will love it. Really helped my PCR's that I couldn't get to amplify well.

Austin