Problems with Geneart Site Directed Mutagenesis - (Jun/15/2011 )
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!
Quickchange kit is good for plasmid bigger than 10kb if your plasmid is less then maybe u can try finnzyme...once people told me, which also dealing his project with this site directed mutagenesis, the temperature playing critical role..try to use many time...maybe also can try to think about chimeric pcr...
keep trying dont lost hope...we need to keep strong
Rubicon how's the result? u still cant get it?? i guess u need to up the annealing temp...some case i ever had with colleagues, the mutation done at 71..we at first not trying this because it really rare people use 71-72C as annealing temperature, however u can consider this too coz ur Tm is very high.....did u ever heard about phosphorylation added primers...i heard it can solve this prob but i am not sure about this...
Decrease the annealing temp to 52-53. Use 400ng of primer (crazy but works), increase the amount of dNTPs. Try extending the 3' end of your primers. Use more of the PCR product + more bacteria for transformation. Very general, but these tips have all gotten me colonies when it seemed futile. hope this helps.