Recombinant PCR - (Jun/18/2008 )
I am stuck up at the recombinant pcr. Let me explain what I am planning to do... I am amplifying two gene products with a connecting element between the two. My primers are such that one primer have the connecting element (that is the reverse of first amplicon and the forward of the second amplicon). After this, I am planning to combine two genes in the pcr reaction. I could get the first two pcr reactions properly, but I am not able to do combine them both. I am getting lots of nonspecific and no specific amplification at all... I tried all the methods but of no use. Can anyone please suggest a proper method in doing this. I am getting little desperate now.
I know what you're doing, I do it rountinely and it generally works quite well. If you could give your primer sequences and your protocol for the second round of PCR (where you try to combine the two products to make a new longer one) I can try and help troubleshoot.