I want to create mutated DNA by using quick change site direct mutagenesis. Quick change works by using a pair of complementary primers with a mutation. In round of PCR cycles these primers anneal to the template DNA, replicate the plasmid DNA with mutation. The mutant DNA product has a strand break (nick). I am following agilent technology protocol (attached) it says to use XL1 blue super competent cells to repair nicks. Does anyone know if I can transform them into DH5alpha? Will that nicks will be repaired or not because some genes such as RecA have been mutated in DH5alpha?
I also want to know following points:-
1. Instead of Pfu turbo DNA pol enzyme (As in Agilent technology kit) here I am using Q5 pol is this ok ?
2.How to set annealing temperature for mutagenesis primer? (Primer sheet with PCR cycle is attached).
Previously I have performed mutagenesis but no able to create mutated DNA. I used 10 ng DNA and same primer as mentioned above.
Primer for quick change site directed mutagenesis (SDM) are exact complementary to each other so I set up 3 sets of PCR reaction to perform SDM.
1. only mutated F primer
2.only mutated R primer
3.Both mutated F & R primer simultaneously
After this I have mixed independent F & R PCR amplified sample and digested these mixed and simultaneous F&R primer amplified so called mutated samples by DpnI digestion (to confirm DpnI digestion control was setup and after transformation I did not get colonies for DpnI cut control sample), then 2ul of this DpnI digested sample were transformed in 50ul DH5alfa competent cells.
After transformation I picked colonies of these so called mutated samples and sent it for sequencing but I am not getting mutant.
After mutagenesis my sample is wild type (no change in bp) I need to change only 1 bp so during primer designing I substituted only 1 bp so that I can get change in 1 bp after mutagenesis.
Kindly suggest me how to troubleshoot this ?
Yes, you can use DH5alpha cells for the mutagenesis procedure.
Regards, Christian
It might be better to use the DNA polymerase supplied with the kit (maybe the whole procedure is optimized for the usage of that polymerase). But you get some colonies so that is probably not the problem.
The primer design tool offered by Agilent usually gives good results. You can find it in the attached link.
A question: what kind of bacterial strain did you isolate your template DNA from? I am asking because this strain must be DNA methylation positive for the mutagenesis procedure to work correctly.
I hope this information can help you with your problem.
With best regards,
Christian
http://www.genomics.agilent.com/article.jsp?pageId=7100004&_requestid=37855
Hi Monika,
Quick change is a semi-PCR method. So use more DNA as template (I use 80-100ng).
Then add your F and R primers in separate reactions for 6 cycles. Then after, mix your reactions and run PCR for 24 cycles. Otherwise, your complementary primers anneal and you will find native template at the end of your experiment.
You can use any High Fidelity DNA polymerase ( I used PRIMESTAR from TAKARA and Q5 from NEB and both worked properly).
After treatment with DpnI transform your PCR product to DH5alpha to amplify the plasmid. I have used DH5 strains several times and it was perfect.
Wish you find it helpful.
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