Hi all,
I trying to do single site directed mutagenesis with my 6.4kb plasmid. Initially I designed the primers around 40nt long, but since both the primers are exactly complementary, I was seeing strong primer dimers even with high concentration of template.Then I reduced the primer length to 30 nt, this time around I got visible bands in agarose gel, but hey were always slightly smaller in size then expected and obviously when I transformed them I did not get any products.I used the a modified version of strategene protocol but with Phusion. The conditions I used were,
Initial Denaturation 95 for 5 minutes
Denaratuion at 95 for 50 sec
Annealing at 75 for 1 min
Extension at 68 for 3.5 min
Repeat 20 cycles
Final extension 7 min
I also did a touch down pcr as well starting the annealing at 78 and down to 68 for 15 cycles and then another 15 cycles at 68 annealing temperature. In both I see nice bands but always slightly lower in size, sometimes I suspect it may be around 6 kb and sometimes around 5 kb for my expected size of 6.4kb. I donot see any colonies after transforming to E.coli Dh5alpha at 2500 volts. Anyone has any sugesstions.
Thanks
Hi Gowri,
I recommend our protocol for site-directed mutagenesis. See:
http://www.ncbi.nlm.nih.gov/pubmed/19566935
You can download the reprint from
http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-9-61
According to the paper's citation record, it has been used in many different systems with success.
Best wishes,
Israel
Thanks a lot...But I can see that you have used Pwo polymerase...But will this work with Phusion..
We have not tested Phusion with the protocol.
If you do, I'd greatly appreciate if you inform me.
The best protocol that worked for me during site directed mutagenis using Phusion was inverse PCR placing the mutation on the 5' extremity of the foward primer. It worked everytime.
Have had good success with the Agilent Technologies (formerly Stratagene) Quick Change Kits.
You didn't mention how "severe" the mutagenesis you are trying to do is, and tacking onto Penny E Shockett's answer, if doing multiple base mutations, insertions, or deletions, I used the QuikChange method but with the two-step modification as in the attached 1999 publication + tips/details with very good success. It works so well that I don't think I went back to the standard QuikChange technique for more than a single base change. It specifically deals with the problem that the mutagenesis primers are exactly complementary while the primer-template binding is not. I used Pfu Turbo with this protocol.
Thanks Christopher S Morello. Actually, we've used an adaptation of the two-step modified protocol. See: https://www.researchgate.net/publication/7105810_Slow-inactivation_induced_conformational_change_in_domain_2-segment_6_of_cardiac_Na_channel
Article Slow-inactivation induced conformational change in domain 2-...
hello I'm doing the site-directed mutagenesis using the same method with you. The size of the plasmid is 6 kb and Phusion enzyme. I want to know whether you have succeeded your work eventually?
Hi, I used the protocol suggested by Prof. Hanukoglu using Phusion and it worked phenomenally well and I got positive colonies every time. thanks to the Prof for the suggestion.
I recommend our protocol for site-directed mutagenesis. See:
http://www.ncbi.nlm.nih.gov/pubmed/19566935
You can download the reprint from
http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-9-61
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