Make sure you have a product by running the product on agarose gel, excising and purifying, followed by a blunt end ligation. Also check that your primers a phosphorilated at the 5' end (not sure if quick change requires phosphorylated primers).

I prefer to use phusion polymerase and 5'-phosphorylated primers. I run the PCRs, gel extract the product and then do a blunt end ligation before transformation. I don't trust DpnI too. Also ensure to calculate your annealing temperature using the part of you primers that actually anneal to the template.

Hi Nagendra, 

I haven't used that kit before unfortunately, but I do have a fair amount of experience with site-directed mutagenesis. By the sound of things, the details you have provided about your primers sound fine. I usually use the PCR site-directed mutagenesis method on a plasmid and I have attached my protocol to this message. I start with 200 or 250 ng of plasmid and use Phusion DNA polymerase - I always make sure that the DNA extension time in my PCR cycles is more than enough for the size of the plasmid I am using (>10 minutes). This is to make sure that the polymerase has enough time to amplify the whole plasmid sequence.

Like Rommel has said, you should definitely run an agarose gel after the PCR cycles to ensure that you definitely have products; I always remove the original template from the PCR products using the restriction enzyme Dpn1 (this will just leave the PCR products intact). You will be able to see the digestion of the original template DNA if you run some of the sample on an agarose gel after this step (a control will also show you that the enzyme is working correctly). 

If you can see products on your agarose gel after the Dpn1 digestion, but don't get any bacterial colonies after transformation, it could point to an issue with your competent cells. I use E.coli DH5a cells and usually don't have a problem with them. Alternatively, if you run the site-directed mutagenesis PCR cycles and perform Dpn1 digestion, but afterwards don't see any bands on your agarose gel that are the right size for your plasmid, there's a problem with your PCR reaction. Most of the time when I have problems with SDM, it's usually because of an issue in the PCR cycles. Sometimes I find that adding a little DMSO into the PCR reaction helps - try a final concentration of 3% (1.5 µl in a 50 µl volume), or if that doesn't help then you can go up to 10% (I wouldn't go higher than this). 

Hope this helps - good luck with your experiments! 

What is your PCR program? I used 50-55 Celsius degrees (sometimes you have to decrease temperature for better efficiency) and 1 minute for primer annealing and then 68 Celsius degrees for elongation (try to extend elongation time as much as possible - for 6kb I would use 8 minutes). 27-28 cycles should be okay. I used Pfu ultra polymerase. Try to perform DpnI digestion overnight and then transform DH5alpha cells. Spread all culture after transformation on your plate. Oh, and I used 10 ul of digestion reactions for transformation without running get etc. And it worked in my case.

Good luck :)

Hi. Everyone thank you for sharing the idea.I used it the following (18 Cycle) PCR program and further steps.  

A) 94'C=3 min, 94'C=1min, 52/55/60'C= 1min ,68'C=6 min ,68'C= 60min., 4'C=hold

B) Then Dpn1 digestion 1ul for 1hr at 37'C.

C) Transform 4-6ul Dpn1 digested product+ 50ul DH5 alpha 

D) Plating on LBA & incubated O/N at 37'C

Result---- Zero Colonies

If you aren't getting colonies, it means you have no product. DpnI has done it's job of destroying the template and maybe more. I recommend you query your primer design if that's Ok, then try another polymerase.

You can add DMSO or betaine to improve your PCR reaction. Definitely make more PCR cycles - even 30. Try to use different polymerases. As I said - for me Pfu ultra worked fine. Sometimes I wasn't able to see my PCR product on the gel, but after transformation I had some colonies with mutated site of interest. Good luck 

Hi ! 

I had the same problem, no colonie obtained when I used 50 or 100ng of plasmid, but it worked (100 < colonies < 150) when I have tried with 150ng.

Good luck 

Hi Nagendra,

I have used this SDM kit. Generally, I found that the transformation efficiency rate was very low, although I also had a very low rate of false positives.

I start with 20 ng plasmid DNA. I use PfuUltra HF DNA polymerase and make sure to keep the primers in excess (125 ng of each primer in the reaction, as recommended in the kit protocol).

I had a particularly problematic mutation and I tried increasing the amount of DNA, playing around with PCR cycles and temperature etc..., but I found that slightly moving the primers did the trick. So you could take another look at your primer design - the desired mutation should be roughly in the middle of the primer sequence.

Good luck!

Hi. everyone.thank you so much for sharing your nice experience.Fortunately, I've got 27 colonies using PfuUltra (2U) along with 50ng (6kb) plasmid. I sent the sample for sequencing. Let's see really mutation will be incorporated in those colonies or they are just false positive colonies.