your experience with long range PCR - (Aug/30/2007 )
Hi, if anyone has consistently produced a variety of ~ 10 kb sized amplicons from human genomic DNA, could you share your experience here? I'm probably going to generate overlapping 10 kb products on a high throughput format for sequencing in the near future.
-enzymes/ kits especially the newer solely proof-reading enzyme formulations such as iProof, Phusion and Stratagene Pfu series
-relative ease of optimization (or rather minimal optimization required)
As you know, be very cautious in your template selection. For example, don't expect to be able to amplify 10kb from paraffin...
I've used a kit from Takara to amplify 8kb and got nice results in terms of yield and fidelity.
One of my colleagues published a paper:
Long-range (17.7 kb) allele-specific polymerase chain reaction method for direct haplotyping of R117H and IVS-8 mutations of the cystic fibrosis transmembrane regulator gene.
Pont-Kingdon G, Jama M, Miller C, Millson A, Lyon E.
J Mol Diagn. 2004 Aug;6(3):264-70.
Check it out. It may provide you with what you need.
My template source is from blood or lymphoblastoid cell line, so there should be relatively free of inhibitors.
i use LA Taq from taqara, my DNA is human genomic (from blood), and i have very good results until 9kb following the instructions of the kit.
i want to go up to 30kb. i havent tryied yet.
if you need more info tell me.