How to amplify large fragment by PCR
Posted 24 November 2004 - 11:34 AM
If you have experiences on amplification of large DNA fragment, would you like to share with me? I appreciate your help.
Posted 25 November 2004 - 12:50 AM
Edited by arwen76, 25 November 2004 - 02:03 AM.
Posted 25 November 2004 - 10:00 AM
So you mean to use a combination of Pfu and Taq? How about your expanding time? 1kb/ 2minutes or 1kb/minute? I set the expanding time 8minutes for my 6kb fragment, but I got a clear specific band around 2kb. Does it mean that the expanding time is not enough?
I will try the combination of these two polymerase, hopefully it works well:)
Posted 28 November 2004 - 10:15 AM
Make a normal PCR protocol with an apropriate elongation time, then programm it so that each cycle the elongation time is incresased by 5-20 s.
In addition you can use the mix that arwen76 proposed.
It is often called elongase (or similiar) and you can get it from about any polymerase manufacturer.
If you need higher fidelity as obtained from Taq there are also proof reading polymerases around which are usable for long PCRs (e.g. KOD from Novagen).
Posted 02 December 2004 - 09:35 AM
my advice is check your genomic DNA quality and select potent polymerase.