I am doing Site directed mutagenesis using QuikChange™ site-directed mutagenesis kit. The primers designed and protocol followed were as per "An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol" (link shared below). After SDM-PCR, amplified plasmid bands were visible, so there is no problem with the primers. Transformation was done directly after DpnI treatment. In the same transformation the plasmid without mutation showed numerous colonies, so there isn't any problem with the transformation as well [competent cells-BL21(DE3)].
- Tried transformation in XL-1 Blue super competent cells and expression host- BL21(DE3).
- There is no problem with antibiotic used.
- Double-checked the primers, PCR program and transformation procedure, but no significant problem could be found.
Link : https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91
Hi,
What do you mean with amplified plasmid bands were visible? how did you tested this? could you please provide more info in this part?
After Site Directed Mutagenesis- PCR, I ran PCR products on agarose in which two bands were visible: upper one was nicked plasmid (the amplified plasmid band I referred above) and the lower one was of template (circular plasmid).
Hi@Shweta Kishen,
I recommend you to go for sequencing using the PCR product you got after mutagenesis to confirm your target.
purify your target by electrophoresis carefully on a cold buffer to use for transformation.
you can slightly decrease or increase the time for the heat shock of your component cells.
Use a positive control at the same time to be sure that your cells are viable.
use a different plasmid if applicable at the same time such as pENTR 4 or pcDNA 3.4.
Be sure that your medium is fresh and of a suitable temperature at the time you culture your competent cells.
I know that cloning needs patience.
Good luck
- Badges
- Science method