I am already getting crazy with this mutagenisis... I see that most of you are using kit from Stratagene, however I decided to order one from Invitrogen - Geneart seria.
I desinged my primers to get mutated site in the middle... the Tm is about 80, number of base pairs - 41.
Tried already several conditions for PCR: incresead - decreased the amount of template from 20 to 100ng, changed annealing temperature from 55 to 62, used different amount of polymerase 4-20 Units, added DMSO to prevent dimers... nothing helped... no idea what to do next...
I read somewhere that primers should be HPLC cleaned then PCR works better, could it be the case?
the most intriguing thing is when I load gel with PCR products I always load one more well with the same amount of template as a kind of positive control and the thing is that I see band on around 5kb only in the case of positive control... what happens with the template which I use for PCR? why I dont see it on the gel if the amount is the same?
Any ideas what to try next?
Thanks in advance!
Edited by Rubicon, 14 June 2011 - 11:41 PM.