Hi everyone!
I want to make a single-point mutation by quick-change PCR. my DNA fragment is near to 11Kb and is cloned with the Gateway procedure. I got a single band in gel agarose upper 10kb region with split PCR and pfu (forward primer in a tube and reverse primer in another tube, after 5 cycles I add them together. the annealing temperature is 57°C- the second PCR cycle is 11 (the total cycle is 16 ). I think the size of the PCR product is ok. after that I add Dpn1, Then it is transformed into DH5 alpha at 28 °C (Do u have any recommendations about transformation in Sure2 competent cells for large plasmid size?). unfortunately, after plasmid extraction, I see a bigger or smaller size than my native plasmid. I did digestion with proper enzyme but I saw different sizes compared to native. I think I have DNA recombination in bacteria when I transform ( because I am not sure if I also need to add ligase before transformation? I know there make a nick in the plasmid after quick change PCR). Do have any suggestions for my procedure?
Best wishes,
According to the original protocol of quick change you don't need to add a ligase.
Try to linearize the extracted plasmids with just one enzyme and see what happens (if it is the expected size ).
It seems you are using the by separate PCR method, you would try 11 cycles for each primer reaction and then 20 cycles else (total 31 cycles).
In case you want to try other method , there is other call Q5 mutagenesis. You don't need to buy the kit (from NEB) you can use pfu, phusion for ligation a regular T4 ligase and for removing template DpnI, but you need to design new primers .
https://international.neb.com/faqs/2013/01/28/how-do-i-design-primers-to-use-with-the-q5-site-directed-mutagenesis-kit
Noe
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