The Stratagene kit is pretty good.
Alternatively you can use hybrid PCR to incorporate the mutation by overlap PCR and clone that fragment in your plasmid of interest.
Thanks , Koen! Are you familiar with Q5 mutagenesis kit? Do you know what makes pcr-based mutagenesis 'better' than quick change?
The overlap PCR method can be done for a short fragment that then can be subcloned, while the typical mutagenesis kits amplify around the entire plasmid.
For overlap PCR, you do two PCRs that have overlap in the section to be mutagenized, and then you can fuse them together in a secondary PCR. You can generate easily point mutations, deletions, insertions, ..., like this.
I agree with Agata. I did mutation and I got very good results with this method.
Thanks for your help! Hopefully, this time quick change will work.
For another project, I need to make > 20 mutations for cysteine labelling. So, do you guys think the trick in quick change is in the primers design step?
Let me correct what Koen Venken said. Overlap PCR can also be used to create site directed mutagenesis, insertions, deletions, etc. It's a fantastic method that when used properly simplifies your molecular biology needs.
I've been using the QuikChange method for at least 15 years, rarely buying the actual kit (like Agata) but buying the polymerase (PfuUltra) and super competent cells (XL1blue) from Stratagene (now Agilent) and the rest of the components from wherever is best value. I also have used overlap PCR, and find QuikChange best for missense, deletion, small insertion. Note that QuikChange is not really a PCR method- parent DNA only is the template for each round of synthesis… Now that there are modifications to use long templates (XL) and to decrease the time to mutation (Lightning), for me this is usually the way to mutagenize.
Thanks a lot! I will try pfu and Q5 pcr kit on the remaining mutations (>20) and I will share the statistics !
I usually use Quick Change Lightning kit (I also did Multi-site kit). All work very well. I have done insertions of up to 22bp and deletions of up to 500bp.
One very important thing is to use very little plasmid as template in your PCR reaction. I usually use ~50-100 nanograms of plasmid in 50 microliter reaction (usually 0.1 to 0.2microliter of my plasmid prep). If you use a lot than not all the original (non-mutated) DNA can be digested by DpnI digestion after PCR. This causes to get a lot of colonies containing non-mutated DNA on your plates.
For transformation use highly competent cells if you use the reaction directly for transformation as in the manual.
Good luck with your mutagenesis!
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