I am trying to get a nearly 3kb fragment with Phusion (from NEB). The reaction was set up as follows:
1) 5X reaction buffer (I tried both HF and GC buffer) 10 µl,
2) dNTP 1 µl (Final concentration 200 µM),
3) Forward primer (designated F1) 1 µl (making final concentration as 1 µM),
4) Reverse primer (designated R1) 1 µl (making final concentration as 1 µM),
5) Template DNA (from cDNA) 2 µl
6) DMSO 1.5 µl (making final concentration as 3%)
7) Phusion Pol 0.5 µl
8) Di-water up to 50 µl
The reaction condition is as follows:
Initial Denaturation 98°C (2min30s); 98°C (10s); 72°C (25s); 72°C (3min); Final Extension 72°C(10min)
I am sure my template does not have any problem, because I successfully got the correct fragments by using the primer F1 and another reverse primer R2 (about 1.2kb), as well as by using another forward primer F2 and the primer R1 (about 300bp). Also, the reason why I used 2min30s to initiate the denaturation is that I think my cDNA sample is a complex mixture. You may wondering why I use 72°C as the annealing temperature. Because I used the NEB Tm calculator online, and it gave me this answer. This annealing temperature also worked well with primer pair F1&R2.
The only different between the F1&R1 product and F1&R2 product is that the former is more than twice the size of latter. Why I already increased the extension time but was not able to get anything but some small size primer dimers. I check the primer dimer score of F1&R1 and it was not worse than F1&R2 and F2&R1. What happened to my Phusion PCR???
I appreciate your help!