I have been attempting Site Directed Mutagenesis on my gene of interest cloned in the vector pET28A+. I began with two mutations, did PCR and transformed the PCR products into BL21DE3 cells. After several repeats, colonies have appeared on my agar plates, albeit small and opaque. They were cultured but the plasmid DNA isolated from the cells are appearing as smears on the agarose gel. I have decided to try colony PCR to get further leads. How should I design primers for the colony PCR? Any suggestion shall be highly appreciated.
colony PCR does not require any different type of Primers, primers u have designed for amplifying ur specific gene can be used , only instead of template u have to add a bit of ur transformed colony and run the PCR reaction......
he is right.
It is not requisite to design new primers :)
you can use your created primers
Although this is not about colony PCR, I believe it's very relevant to the issues you currently have.
You should NOT use BL21(DE3) for cloning purposes, because the strain is recA+.
You should use recA- strains like XL1-blue or DH5alpha for cloning procedures. Once you are sure about your expression construct, transfer the plasmid to BL21(DE3) for protein expression.
Thank you all. I have previously performed transformation in BL21DE3 with the WT of my protein of interest, the gene being cloned in pET28A+, and it was a successful one. However, as I tried doing SDM, I'm not getting positive results.
Hi @Subhoja Chakraborty...
For the colony PCR, just like it has been pointed out you can use the same primers you had employed in amplifying your GOI. There is no reason to deign a new pair of primers since the aim is to confirm the presence of the GOI.
And as for the smearing you experienced with running the plasmid on agarose gel, (if the smears are light) make sure that you load enough sample or that the concentration of the plasmid is sufficiently high enough as to be detectable on the gel after electrophoresis...
While you can use your GOI primers for colony PCR, you can also confirm via sequencing with Universal primers. Depending on where you cloned your GOI, you may be able to use the T7 primers within the pET28A+ vector. You do not have to buy these primers; sequencing companies can add them to your isolated plasmid (like genewiz) for your when you send it for sequencing.
https://www.addgene.org/vector-database/2565/
I am not paid by genewiz to advertise their services, but I do utilize them.
You can learn more about their Universal primers and sequencing requirements in the links below:
https://www.genewiz.com/Public/Resources/Sample-Submission-Guidelines/Sanger-Sequencing-Sample-Submission-Guidelines/Bacteria-Phage-and-bacs#sanger-sequence
https://www.genewiz.com/Public/Resources/Free-Universal-Primers
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