I have 7.0 kb plasmid (with the insert). I performed Site directed mutagenesis and I have nice colonies grown in a plate (km). Now how do I confirm from the colonies that they contain the desired mutation. I have designed primers on a region (parental vector) that would contain mutation. So my question is, can I still use primers designed on parental strand to verify the mutation on mutated strand or vector. If I do a pcr using these primers then what would it look like on a gel?
I do not think that you can easily distiguish between wt and point mutated gene by pcr. In my experience the clones you pick are positive for the mutation. Usually i would pick 3 and send them for sequencing
You can't be certain unless you send it for sequencing. To have a higher chance of success, treat your PCR product with DpnI (just add 1 ul) to get rid of the parental DNA. Hopefully you will only have mutated DNA in your colonies.
Thank you Alexandros. since I did not have X-gal and IPTG on my kanamycin plates, i was uncertain about my colonies so i wanted to make sure something prior to sequencing. Now i will send 3 or 4 colonies for sequencing. But my concern is during my earlier transformation experiments I did not use x-gal and IPTG, and still worked, but I am concerned if that works for mutagenesis experiments as well.
Kanamycin plates should be ok. I don't use X-Gal and IPTG and I get mutant colonies (usually 1 out 3).
That's great news for me Alex. Thank you so much for these for informations.
Dear Bidur Paudel,
Unless you have sequenced your DNA, you can't predict if your site directed mutagenesis experiment was successful.
There are a few exceptions:
1) If your mutagenesis results in a new and unique restriction site in your construct, you could perform an analytical digestion after plasmid preparation from the single colonies. You would expect a unique pattern for the mutated constructs versus the non-mutated constructs. Of course only after removing your template from the PCR mixture with DpnI as mentioned by Alexandros.
2) If your site-directed mutagenesis would result in a known color change of a e.g. GFP or other related fluorescent enzymes, a test expression could confirm successful mutagenesis as well, since the codon changes are directly linked to known protein properties.
Sabrina
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