I am using Q5® Site-Directed Mutagenesis Kit by NEB. after performing KLD reaction for 5 minutes at room temperature I went for transformation using the high-efficiency competent cells provided in the kit. I am getting no colonies on plate. Any suggestions.
It is likely that your PCR did not work (well). This may happen if the reaction/cycling conditions are too stringent (annealing temperature too high, too little template..). You can increase the amount of your plasmid, and run a couple of PCRs with different annealing temperatures to determine the optimal one.
For transformation into the chemically competent cells there is no need to increase the DNA concentration - you can simply transform the entire reaction. Also, gel extraction can be somewhat inefficient, actually leading to lower DNA concentration. Since you obtain no colonies, I suppose your enzymes in the KLD mix are fine (DpnI likely to be most sensitive one).
The inability to obtain colonies (provided that everything is alright with the mutagenesis itself) might be due to: large plasmid size (lowers transformation efficiency), toxicity of the insert, etc.
Totally agreed with Ana Crnkovic : Omit the gel extraction step! In most cases, it is a sure solution to ruin your DNA and all follow-up reactions. If you see a clear product at the right size after PCR, everything should be fine, proceed to the next step - or use a column kit (no gel) for purification of your PCR. The kit relies on non-overlapping primers, exponential amplification - PCR should yield high product amounts. The ligation / DpnI step should work fine afterwards.
I am getting multiple bands after PCR. That is why I had to go for gel elution Johannes Stuttmann
You do a PCR on plasmid, so your template is not a complex mixture or anything alike. You should get one major product. A bit of by-product is not an issue; you do not have to make a gel elution for that. Better a few additional false guys than nothing at all.
Post an image, if your problem still persists.
Jayashree Niharika Did you ever get this to work? I am having similar issues. I tried a gradient PCR with varying concentrations of template and cycle numbers and the best PCR product I could get was a mixture of sizes so I tried to gel purify the fragment and proceeded with KLD treatment. I check the KLD product by PCR with vector specific primers (not the same ones used for mutagenesis) but there was no product whatsoever. I have attached an image of my initial PCR products for reference.
Hi,
It is important to include a no-Polymerase control. This will allow you to unambigiously decide whether you do have an amplicon, or whether these bands are just corresponding to your plasmid template. I wrote an SDM protocol at some point for a student
https://www.protocols.io/view/site-directed-mutagenesis-behgjb3w
If you download the pdf, there are some example images for the PCR results. You see that there is a quite strong signal for the PCR product (18 cycles). With the image that you just uploaded, I would have doubts that you actually amplified something.
Bests,
Johannes
Gel elution of your amplicon is not necessary. We routinly do SDM using PCR with overlaping primers (primers have specific SDM) for 18-22 cycles followed by Dpn1 digestion for 2-3 hours. The important thing is that your template plasmid concentration should not be more than 100 ng (40-50 ng is ideal). Try this.
Good luck,
Kishor
Dear Herman,
I do not observe any change in the growth of colonies. After 14 hrs of plating, I proceed to check with PCR. I generally do SDM to mutate internal RE sites hence after mutagenesis, I check the RE digestion pattern compared to WT. You can also confirm the mutation by sequencing.
Regards,
Kishor
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