quikchange question - (Nov/06/2007 )
Hi Fellow researchers,
I've been trying to perform a quick change insertion/deletion (removal of 3 insert 4). I ran my reaction using the phusion high fidelity DNA polymerase. However, I'm not getting any colonies after transfomation onto amp plates (the plasmid are ampR). I set up the reaction based on the
initial den 98C 30s
den 98C 10s
annealing 55C 30s (the 55C is from the quikchange protocol)
ext 72 2 min
final ext 72C 5min
I also ran a gel of the reaction tube after PCR and detected no products (which I understand is common due to the low concentration)
Any insight would be great
Have you checked your primer for annealing at 55°C? Go to http://www.stratagene.com/QPCR/tmCalc.aspx and calculate + follow the instructions of the quickchange manual.
Apart from this: for Pfu it has been shown that at 72°C they have strand displacement activity which @ 68°C it has not. I'm not sure about phusion polymerase, but maybe you need also to lower your extension temp a bit? (this would not result in not having colonies at all, rather in having colonies without the desired changes, but this could save you a lot of work once you get the reaction to work).
One hint I can give you: carefully read the quickchange manual and the faq's on stratagene's website (and then go ahead with your own materials without buying the kit).
After you preform the QUIK change pcr, do you need to pruify the reaction tube or can you directly transform into TOP10 cells directly from the PCR reaction? I'm asking because I ran a gel of the PCR and it was not clean (smeared bands)
After PCR I do DpnI digestion (stratagene recommends this, the phusion polymerase says it's not necessary), and then I just transform my competent cells from this mixture.
At 18 cycles you wont see too much on gel, If you're not sure about PCR conditions: run a pcr for 35-40 cycles and put it on gel, later perform the same PCR but then for 18-25 cycles for your actual mutagenesis.
Now I'm a bit confused as to what is the correct protocol for the QUIK change. I know you start with the PCR cycles. After that, do you run a gel ( to verify) or add dpn1 1st then run a gel after the digestion. After addition of dpn1, do you need to column purify the pcr product before transformation?
We digest with dpn 1 and then transform cells without any purification. One thing could be that the enzyme is not amplifying the plasmid, which could be due to secondary structures. In this case, try adding some DMSO to the reaction. It could help.