I use human cDNA library as a template (100ng)
I have changed the buffer, which didn't change anything.
I use 2 min 30 sec initial denaturation time 98 C as I suppose human cDNA counts as a "complex" template
denaturation is 30 sec at 98 C
annealing 20 sec at 63 C
extension 40 sec at 72 C
and final extension 10 min at 72 C
I have exactly that one 1 kb band and everything looks perfect... then I do gel extraction, digestion, purifying, ligation, transformation, plasmid purifying, digestion to see if I have the right insert... and my 1kb bands are looking fabulous... then I sent them for sequencing and receive back some nonesense. I switched from Taq to phusion because Taq made a few mistakes... but this phusion... it's not doing it's job or maybe I really can't use it right Others get fabulous results with it and me... has anyone ever worked with human cDNA libary and a phusion enzyme? Is there some kind of special tips how to make this working... in one of the samples the phusion had already a gap in the third nucleotide in the primer... so it would seem that the annealing time is too short? But then again... the gaps are random. Sometimes in the middle, sometimes in the end and sometimes in the beginning... I have double checked the reactions to make sure that I didn't have any contaminations... I have tried everything... maybe there is something wrong with the cDNA libary....
Edited by Juliasarmoire, 29 October 2009 - 06:40 AM.