I usually use the Quik Change method using Pfu and complementary oligos. The Phusion (NEB) protocol says to not have overlapping oligos; they should be phosphorylated and then the PCR product is ligated. I found an alternative protocol that says the oligos should overlap but have about 8 bases non-overlapping at the 3' end, treat the PCR reaction with DpnI and transform (ie. just like Quik Change but the oligos are different). I essentially want to use the Quik Change method with Pfusion instead of Pfu, so my oligos are designed as completely overlapping. Has anyone done this successfully with Pfusion? Please provide brief protocol for thermal cycling.
Hi,
The polymerases Phusion and Pfu are both fine but the main difference is this:
- if you use completely overlapping oligos youur amplification is linear (not a proper PCR) and not exponential (each strand generated by primer F cannot be used as template from primer R and vice versa). This means you need to start with a lot of template plasmid and then it is a must to use DpnI to get rid of this strong backgroung.
- if you use oligos non overlapping (and ligation at the end) at all and oriented "tail-to-tail" then your amplification is exponential instead (each strand generated by primer F is the template for a new strand generated by primer R. Only 1 primer needs the mutation) so you need a little of template plasmid and may not even use DpnI since the most of your product will be non-methylated (being the amplification exponential - i.e. a proper PCR).
Max.
the protocol will depend on the Tm of the oligos....but I have performed this successfully.
I've done it with Phusion and it worked fine. You can use primers as described in the QuikChange protocol. For the PCR program it always depends on your template... usually it works fine with the recommended 50°C. For the rest use the parameters of the Phusion manual. You may get a better result (more PCR product) if you prolong the extension time a bit (use the full 30s/kb).
In any case make sure to get a complete DpnI digestion - 3h at 37°C to overnight incubation is no mistake.
good luck!
Phusion is awesome (way better than Pfu), and your protocol will work fine. If you're concerned about matching up melting temps, just plug your plasmid sequence into my restriction free cloning web-tool, mark the base you want changed, and it'll pick your primers for you (www.rf-cloning.org). You can check out the protocol I use in the Q&A, but it's essentially a canonical Quik-change.
Good luck!
-Steve
I have successfully used Phusion for mutagenesis. As mentioned, the protocol depends on the Tm (and the length of the plasmid), but I use the following protocol:
95 C for 1 min followed by 16 cycles of 95 C for 1 min, 50 C for 1.5 min and 68 C for 15 min (this time will depend on the length of your plasmid). If you have problems with amplification you might want to try adding DMSO or switch the Phusion buffer (HF vs GC).
The protocol will work fine with Phusion, and you should get more product. I would use the 30s/kb extension that was previously mentioned, but perhaps do 12 cycles instead of the more typical 16. This cuts back on side products.
Phusion tends to work better for me on quikchange mutagenesis. I like it since it's so fast (15s/kb). I don't like the Agilent approach of designing perfectly complementary primers because for successful insertion of your mutation you're essentially gunning for a mis-priming event for your primers to anneal to your template rather than themselves.
Similar to your modified protocol, I like to treat the situation as two separate PCRs. The 3'end has primers that are complimentary to the template at some favorable Tm(65C by NetPrimer). Then the 5' end are overhangs that include your modification and sequence that is complementary to the other primer. What's different is that the Total Overlap Tm should be comparable to the Tm of the 3'end of the primer rather than being completely complementary. This way you're kind of biasing your primers to amplify with overhangs that will anneal to each other after cycle 2. Everything else is the same and you can check for success by looking for a band corresponding to linearized product.
Standard NEB Phusion mixing protocol with HF buffer and 5% DMSO v/v
1 - 98C - 5min
2 - 98C - 30s
3 - Annealing - (50 - 65) - 45s
4 - Extension - min 45s for
No problem at all. Just go ahead do it! It always works fine. For my experience, as long as you get the product with correct size, 99% you succeed! DpnI will remove the background but is not necessary if you try more than 25 cycles and you don't care pick up more than one clone for sequencing. I will prefer DpnI digested over night.
Dear Michael,
Yes, this enzyme work very well as pfu. NEB made a little change in the Stratagene's "Quick Change" method to bypass their patent on the method.
Hi,
The polymerases Phusion and Pfu are both fine but the main difference is this:
- if you use completely overlapping oligos youur amplification is linear (not a proper PCR) and not exponential (each strand generated by primer F cannot be used as template from primer R and vice versa). This means you need to start with a lot of template plasmid and then it is a must to use DpnI to get rid of this strong backgroung.
- if you use oligos non overlapping (and ligation at the end) at all and oriented "tail-to-tail" then your amplification is exponential instead (each strand generated by primer F is the template for a new strand generated by primer R. Only 1 primer needs the mutation) so you need a little of template plasmid and may not even use DpnI since the most of your product will be non-methylated (being the amplification exponential - i.e. a proper PCR).
Max.
I am sorry Massimiliano. I don’t share your opinion. I think it is exponential in both cases. Just draw it down with 5’ to 3’ in each sense. I think whether the oligos overhang or not at their ends, it will only affect the distance between the two nicks left on the plasmid after amplification, which may affect plasmid circularity. Linear plasmids have low efficiency of transformation compared to circular, hence NEB advises to ligate but I don’t think it will be necessary.
In the case of SDM, this is indeed not a real amplification (or exponential increase), just a copy of the whole plasmid, as Massimiliano said. this is why it's better to start with a higher amount of the template. If you use non overlapping oligos and an "inverse PCR" then yes, it's a real PCR. BUt you need to phosphorylate the primers or the PCR product, ligate then transform.
In the case of SMD, no need for phosphorylation since the plasmid will self recycle with a 30 base (usually the size of both overlapping primers) complementarity which is largely enough (don't forget that lambda phage can do the same with only the 12 bases COS, and I myself used this kind of constructs often enough recently to know that it's pretty efficient)
the SMD low number of cycle was originally made to minimise the probability of mutation during the elongation svep by the polymerase and the original protocole recommended 12-13 cycles only.
I used both methods successfully.
I use Natasha PCR condition for Turbo enzyme. For Fusion enzyme, I agree David's protocols. The overhang end oligos are better than completely overlapped oligos. Also you can use I proof Taq from Bio-rad, which is similar to Phusion.
I thinkk I misunderstood the problem! Sorry Massimiliano! You are right!
I use a two stage Pcr for site directed mutagenesis and have done it successfully with high fidelity polymerases from stratagene and clontech. What I usually do is that I set 2 Pcr tubes one containing the reaction with forward primer and the second having the reverse primer only to make ssDNA, After 16 cycles I transfer 25ul from each to a new tube, add 0.5ul of polymerase and give it another run for further 12 cycles to make dsDNA followed by DPN 1 digestion and finally transformation. It always works for me.
The fact that the primers are completely complementary does not matter so much, I have used the Agilent Quikchange mutagenesis kit several times following their exact protocol and it has worked fine, you obtain a very small amount of product in the end but it is plenty for a transformation. I have not used Phusion polymerase though, always used Pfu ultra, Pfu turbo or Pfu lightning.
Abdelhalim it is ok. Discussing thing is always a good stuff for everybody.
Also sometimes I am wrong actually.
Max.
I did this exactly as you describe, with 100% complimentary primers and it worked. Very ugly gels, but I chose the best results for further studies based on the controls I used. Try to keep the cycle times as low as possible for the higher temps to avoid denaturation of the polymerase to soon.
Hello, I was wondering if you had any luck with Phusion along with the perfectly complementary oligoes? Actually I am also planing to do it, so am curious to know what others have got!
I hava a similar issue. There is an article comparing high-fidelity DNA polymerases and suggests that Phusion cannot amplify plasmids with complementary primers efficiently compared to Pfu-turbo. I have at least 5 mutations to generate. Would you recommend to order new partially-overlapping primers to be used with Phusion or to order more Pfu-turbo? Which way is cost-effective or efficient?
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333370/
If I use the Phusion instead of the Pfu in this protocol (with complimentary primers) should I do the ligation after the Dpn1 step or the product will already be circular?
Currently, the exact mechanism of action of the Quik Change SDM method (using fully overlapping primers) is under discussion, with both linear amplification to give a circular product and exponential amplification (true PCR) to give a linear product with short homologous ends being suggested. In both cases, a ligation step would not be required as in the former, the product is already circular, while in the latter, the homologous ends allow for in-vivo recombination and circularisation.
We have greater success with Phusion with the inverse PCR approach (non-overlapping primers) than with the Quik Change SDM method (using fully overlapping primers). Some authors have suggested this to be related to the higher annealing temperature for Phusion PCR and formation of primer dimers.
For a description of the inverse PCR method see:
https://www.researchgate.net/publication/317225742_Inverse_PCR_for_Point_Mutation_Introduction
For a discussion of the mechanism of the Quik Change method see:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333370/
Chapter Inverse PCR for Point Mutation Introduction
Muhammad Adnan I want to try your single primer 2 step mutagenesis approach because all other methods have not worked for my large ~10.5kb plasmid. Can you please give me details of this protocol: which enzyme you used, the concentrations of the PCR components (plasmid, primer, which buffer) and the PCR conditions?
Thank you.
Hi Ishita Jain , please read and follow the method described by Edelheit et al., 2009. I have used this method to introduce site-directed mutations easily in several genes. I suggest first sub-cloning your gene of interest into small size vector approx 3 kb in size and later after introducing the mutation, you can further sub-clone into your final destination vector.
Good Luck!!
Hi Saugat Pokhrel that is the same paper I am looking at. Did you also use Pwo enzyme? Have you tried it with Pfu or Phusion?
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