Hi Monika. You can use two-step technique detailed in the attached paper and supplement to avoid that problem while still using the same QuikChange kit or Pfu Turbo, DpnI, and competent cells that you have. It is so little extra work that I always used this technique instead of standard QuikChange for anything but a single point mutation.

Good luck,

Chris

If you are using quickchange from agilent, then you probably don't need a primer pair. Just one primer (F or R) will do the trick. Strand doubling happens in the special cells that they supply with the kit. All info are in the Quickchange manual.

If you choose to use both F and R primers, it should still be ok. Even if 1/3rd of your primers annealed to parent DNA, your mutagenesis will still work. 

Anyways, remember to use the cells that comes with the kit for transformation. It wont work otherwise.

Thanks for reply, Rajasekar....

Hi Monika. You can use two-step technique detailed in the attached paper and supplement to avoid that problem while still using the same QuikChange kit or Pfu Turbo, DpnI, and competent cells that you have. It is so little extra work that I always used this technique instead of standard QuikChange for anything but a single point mutation.

Good luck,

Chris

Thank you so, much Morello..

I got the concept of 2 step Quick change, but I have a doubt at second step, where I should use 25ul of each primer pair extension reaction  were mixed in one tube with 1 U of pfu DNA pol and subjected to the standard QCM PCR protocol.  

According to the Stratagene QCM protocol, they are adding all things like buffer, PRIMER, Template DNA, DNTPs, etc SO IS IT NECESSARY TO ADD MUTAGENESIS PRIMER IN THE SECOND STEP ?  

IF YES SO AT THIS STEP HOW IT WILL PREVENT PRIMER DIMER FORMATION?

Hi Monika.

First, it's not necessary to add mutagenesis primer in the second step. What may be confusing is that all of the components are added in the first step, and yet they added another 1 U of Pfu DNA pol (I'd recommend using Pfu Turbo) for the second step. I don't know if that extra polymerase is necessary, but my colleagues and I would always add it so we could get our mutants and run with them. I think the rationale might be that this protocol adds some extra cycles to the Qiagen QCM protocol, so perhaps some more enzyme is needed.

Secondly, the primer dimer is not likely completely prevented in the second step. The unused primers from the 2 first step reactions will still be there, but the idea is that the first rounds with the primers separated will generate mutant strands that will then be perfectly complementary to the primers and will then be able to compete better with the primers than did the wild-type plasmid sequence. It is analogous to a touch-down PCR where the initial cycles are very stringent and generate more of the specific product to be amplified in later cycles. There will still be primers binding to their complements in each cycle, but your plasmid will be getting replicated up to a level where it will produce subsequent colonies that will be greater in number than any wild-type plasmid that escapes the DpnI treatment (such as denatured, supercoiled plasmid that is endonuclease resistant).

Several of us in the lab made many mutants using this method that were not otherwise obtainable with the standard QCM protocol, especially when more than 2 mismatches or sequence additions were performed. Many of us would in fact use the Two-Step protocol even when a single base change was being performed just because it was so little extra work. I would do 3 cycles in the first step and my colleague would always do 5, but I think our results were comparable. Lastly, several QCM reactions that yielded no colonies were salvaged by concentrating the DNA and using the remainder in a single transformation as described in the second attached file. That usually yielded only 1 to 5 colonies, but the correct mutant was among them.

I hope that helps and good luck.

Thank you, Morello, for the elaborated answer.

I want to ask one more thing, what is the another strategy (except, quick change site-directed mutagenesis) to design primer for site-directed mutagenesis if I want to change only one amino acid in my insert DNA.

Is site-directed mutagenesis by inverse PCR (base substitution) a correct choice for creating mutation for only one amino acid in my insert DNA.

I am attaching the file for the SDM by Inverse PCR.

Using inverse PCR would be a good choice, too. Just don't forget to purchase phosphorylated oligos if you're not in the habit of doing that (or phosphorylate them or the PCR produce yourself.)

Thanks Morello for your valuable suggestions.

Every case is different, of course, but we find that in most cloning cases now it is cheaper to have a whole new insert synthesized than to mess with site-directed mutagenesis. For relatively short inserts you can have the whole thing synthesized and receive it in the vector of choice (and it requires no work in your lab, except to send the sequence information to the manufacturer, so you can spend the time on other projects). For longer (more expensive) inserts you can pick suitable restriction sites and just swap out the region of interest in your own construct with a synthesized fragment (minimal, routine work). Our cost analysis includes labour (we have relatively expensive hourly technician or PDF salaries to consider), but it almost always comes out considerably cheaper (US$0.18 per bp for synthesis, base price), faster (time is money!), and without the sequence artifacts (which also eat up more time and money to correct and recheck) that we have seen too often with PCR methods. You should consider this approach. Gene synthesis is the future of cloning, and it is here now.

In my personal experience, following the Stratagene Quikchange protocol for SDM using nothing more than my own high fidelity DNA polymerase and DpnI has always worked without a hitch. Primer dimers have never been a problem.

Gene synthesis is a good alternative as long as an economical source is available.