PCR amplification of large template - (Jul/30/2013 )
I have two primers that will amplify my 8kb construct at a BamH1 site and will add 2 novel restriction sites for ligation of an insert. I was wondering if anyone had any pointers on setting up PCR reactions for large templates, such as length of cycles and components, really any advice would be helpful - because while I am able to easily amplify small inserts, the larger vectors Im having more trouble with. Thanks!
First, how big are your primers and how are they designed? Are they overlapping such as with site-directed mutagenesis or are they the normal forward and reverse with each having a different restriction site to be introduced into the product? For large templates, I would recommend you set up the pcr in a series of optimization buffers. These range in pH from 8.3 to 9.0 with varying concentrations of MgCl2 (15 or 35mM) and varying concentrations of KCl (250 or 750mM) all with 0.1%TX-100. I have a spreadsheet if you are interested and have found I can always get a product in at least one of the buffers. Next, always give at least 2 minutes per kb for something that large and use a Taq polymerase, not a PFU. You've likely got some secondary structure the enzyme needs to contend with so it needs the extra time.