I am doing site directed mutagenesis using stratagene-QuikChange II Site-Directed Mutagenesis Kit. The protocol and primer design I'm following are given in the link below. I am not getting any colony after tranformation with DpnI treated PCR product. Though the band of nicked DNA after SDM-PCR (possibly amplified PCR product) is visible (fig1- band labelled 1). The template band is also visible below it (labelled 2). After digestion with DpnI, nothing is visible on the same level as of amplified PCR product. If SDM is not happening than what band 1 is?
The size of the plasmid used as template is 7.4kbp.
Template used- 10ng.
PCR reaction vol- 50ul, loaded on the gel- 10ul
DpnI- 0.5U at 37ºC for 2hrs.
Link: https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91
I think both the bands you see on your gel are actually your template plasmid, just in different conformations (supercoiled vs. relaxed). So sadly, I don't think your PCR was successful, hence no visible band after DpnI digestion. Maybe you can do a gradient PCR to establish a better annealing temperature. I would also suggest you add a template control to your agarose gels where you load an equal amount of template next to your PCR product. That way you can immediately tell from the gel if your PCR was successful.
Mutagenesis PCR can be really frustrating. Some mutations work on first try, some require tedious optimizations in either PCR temperatures or primer design. To be quite honest, I don't really like the QuikChange protocol and have always had a lot of trouble with it myself. Very often the PCR didn't work or there were primer repeats in my sequenced plasmids.
If your problem persists and you can't seem to fix it even after trying some optimizations, you may ultimately look for and try a different primer design or cloning approach. People have developed a lot of other approaches towards site-directed mutagenesis and there is a lot of literature on the topic. Personally, I have been using the "round the horn" protocol very successfully (you can find it on openwetware, but the site seems to have some issues right now).
Good luck!
-Jan
Dpn1 degrades methylated DNA. Dobert is 100% right. It looks like you're amplifying your parent strand based on the gel. I would check the melting temp of your mutagenesis primers factoring in the mis-paired bases at the site of mutation.
First thing I would do is try a different annealing temp which should be at or slightly below the Tm for your flanking sequences.
In our lab we use a very simple primer design. Mismatched bases at the site of mutation are flanked by sequences each with a melting temp of ~60C with AT pairs = 2C and GC pairs = 4C. Hope this helps. Primers are cheap and quick to make/order. I like to use sigma or eurofins.
Hope this helps!
Hi Shweta, The DNA ladder does not look that great. The bands at the top seems missing. What percentage of agarose gel did you use?
The bands were not clean even before the enzyme digestion.
You might want to load more DNA sample to get stronger or missing DNA bands.
Have you checked the DNA quality (260/280 ratio)?
It would help if you also double-check your agarose gel preparation and the buffer
Hi Shweta, I think the bands you are seeing before Dpn1 digestion may not be your PCR amplified band as it should be a single band and more intense. I think you need to get your PCR conditions and try again.
Although it never worked for me many of my colleagues said to me they got clones with out seeing any PCR product after amplification.
Try increasing the extension time, and use 3 or more annealing temp for successful PCR. If nothing works, try designing a new set of primers with some larger complementary regions.
Hi Shweta,
I would suggest use a two-stage PCR: you run F and R primers in two tubes separately for 15 cycles then pause, open the PCR cycler and combine them in the same tube, continue to run the rest of cycles. Using this method, I always saw strong bands of PCR products on my gel and got a lot of colonies after transformation. So far I have sent 15 colonies in total for sequencing (different mutations) and 14 of them had the right mutation. (I use Pfu polymerase + 100% complementary primers)
Hello He Zhou could you explain a little bit more the protocol. currently I have my 100% complemented primers and used the QuikChange protocol. would the program be the same but just in two different tubes or there is anything else that is different.
thanks
Nicolás Feldman Yes, everything else is the same with QuikChange protocol. You only need to run the first 15-16 cycles (adjustable based on your requirement, I run 30 cycles in total) in two different tubes (to facilitate the binding of primers to the DNA template) then combine them in the same tube to finish the rest of cycles.
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