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cloning of a long gene using cDNA - (Apr/18/2012 )


I need to clone a long gene of approximately 5.5kb.
I extracted total RNA then reverse transcipt to cDNA using a specific reverse primer, then PCR using long Pfu DNA polymerase. with various conditions, with DMSO, different MgCl2, different annealing temperature with no success.
I checked the cDNA by overlapped PCR and it's ok.

Please, any suggestions?

Thanks a lot



I also have almost the same problem. I use Fermentas cDNA premium kit which claims it can synthesize up to 20 kb of cDNA, however I have not been able to get PCR product more than 3500 bp with it.
5.5 kb is also considered quite big. I just have the following suggestions:
- next time synthesize your cDNA immediately after RNA extraction.
- use phusion polymerase instead of pfu

and how did you do overlap PCR? what condition did you follow?