I am to PRC out an 8kbp exon divided into 10 parts. Standard PCR was giving messy sequence so my PI suggested using Expand Long range PCR. This is my first experience in a lab and this technique is a new to me. I followed the protocol that came with it and used the Forward and Reverse Primers as such: 1F,10R ; 1F,5R & 5F,10R; 1F,3R & 4F,6R & 7F,10R (Buffer with Mg and Buffer without Mg at 55C, 60C and 65C).
Total vol : 50microliters
I finally got a strong band for 1F,3R. I still have to PCR the rest of the exon. Any suggestions? Am I doing something wrong?
There is a vial in there labeled 25mM MgCl2 which seems to have no use in the protocol. Could you tell me what that is for?
If I run out of the enzyme, is it okay to use Taq? Has anyone had success with that? I have ordered 2 kits only because the amount of the enzyme is low and I ran out while trying to get bands, while the other solutions are in plenty.
I would really appreciate any help, any advice.