Hi Peter,

I frequently use the QuikChange Lightning kit and usually, I pick two clones and both are correct (works also on long plasmids and for the exchange of multiple bases). I don't know where exactly the differences between the two kits are. Unless the "II"-kit ships with a slow polymerase, my recommendation would be that you try shorter elongation times. The lightning kit requires 30 sec per kilobase, 5 min instead of 15 per cycle should be sufficient. I usually do 18 cycles and put 60 to 100 ng template into the reaction. Secondly, you used Mung Bean Nuclease (digests ssDNA) and still find the parental sequence. You say that the source DNA is both ssDNA and dsDNA, thus at least the parental dsDNA should be left untouched by Mung Bean Nuclease. In your protocol, you did not mention Dpn I digestion, did you do it and simply didn't mention it or is there any specific reason why you did not use Dpn I? (digests DNA with a specific methylation pattern; the parental DNA is usually methylated but the PCR-product is not). I am asking because the lightning kit ships with Dpn I, digestion is complete after 5 min and I have virtually never found the parental sequence. On all other points, your protocol looks fine to me. If nothing helps, you might check the purity of your primers or simply reorder them. I deem it is unlikely the primers cause your trouble, but on one occasion, a commercial supplier had sent me primers, my clonings persistently failed and an analysis revealed that they had a purity of only 35% - new pure primers solved the issue.

Good luck!

Best regards,

John

Hi John,

thank you for your comprehensive reply.

You mentioned Quikchange Lightning - after a brief comparison with Quikchange II I think both kits contain the same PfuI polymerase but the Lightning may contain higher concentration (not stated; for kit "II" it is 2.5U/microliter) - in Lightning-protocol they recommend 30 s/kb for elongation step and in "II"-protocol the recommended elongation step is 1 min/kb. Based on this I think I can reduce elongation time to 10 min... I will also try 18 cycles...

Oh, and yes - I simply didn't mention the DpnI digestion - I always do it (the kit "II" contains it although it is slower - the digestion step should last 1 hour).

And for the primers - how do you check the purity of primers - do you run HPLC analysis? Although I hardly believe the primers are the major cause for the problem - all 4 primers were ordered at the same time (they should be the same batch) and they were HPLC-purified after synthesis...

Thank you and best regards,

Peter

I totally agree with John, reduce extension time to at least 9 minutes (to my knowledge,the longer the plasmid the slower the polymerase, so 9 minutes should be fine. Try lowering your template concentration to 1-10 ng, and be sure to perform DpnI digestion. I peronally recommed cloning into XL10 gold but endonuclease-deficient cells wll be fine. If you think it is an issue, you can also try purify your mutagenesis by standard column purification, this gives me zero background.

I noticed that I have better success when I purify mutagenesis products (using any PCR purification/concentration kit), and set up DpnI digest with accompanying buffer.

I have been using Qigene QuikChange XL kit for a few years, and sometimes, I do experience the same problem as what you've got. I am not quite sure what triggered that, but if you can get a single mutation, that means your procedure is correct. I suggest you to use the mutant (single mutation) as the template and do the mutagenesis PCR again to see whether you can get the 2nd base changed.

Hi Peter,

My dirty primers mentioned above were ordered from a commercial supplier and were also HPLC-grade. Fortunately, we have an in-house synthesis facility (they made the pure primers then) and generally check the quality of all oligonucleotides by RP-HPLC (this shows if any residual protection groups are present), IEX-HPLC (separation according to number of charges = length) and finally also by LC-MS.

Good luck for your experiments!

Best,

John

I have been using Q5 Site-Directed Mutagenesis Kit from NEB and it works very well in my hands. You can use their online tools to design the primers, you can print the full protocol for the particular primer set and it is fast. I made some modifications in the protocol. 1. In the cycling conditions use 3 minutes as initial denaturation. 2. After PCR check little bit pcr product on a gel to verify the correct size pcr product before proceeding further. 3. If you are not getting any pcr product repeat the PCR with one degree lower annealing temperature.

Hi Peter, 

Looks like you've got plenty of good information but I'll just chime in to say that many years ago, I had lots of success with the Quikchange kit. Then after not using it for a long time, had to do some more mutagenesis. Got the usual kit and I just could not get it to work.. tried multiple primer sets and other things.. it was driving me insane because it used to be easy and simple in my hands.

Anyway, I switched to NEB Q5, got primers according to their design, and it worked immediately.

Good luck!

I am grateful for all the answers full of useful information.

Especially thanks to AnthonyKi Lui for a very interesting suggestion. Luckily my second pair of primers (for double base pair change) does anneal to the mutant with additional base pair (one more) compared to original template. It took me some time to repeat all the work using a mutant as a template and to sequence few clones and I am glad to say that it worked! I've got the mutant with two bp changes.

So, thank you again, Anthony and good luck!