I am trying an experiment in which I measure the NHEJ efficiency of a mutant yeast strain and compare to wild-type to analyze whether the mutation affects the NHEJ process. I take a low copy number plasmid, digest it with restriction enzymes creating sticky or blunt ends and transform them (as well as the uncut plasmid as control) into mutant and wild type strains and plate them onto selective media and count the number of colonies after 2-3 days. I'm wondering if CIP treatment of digested plasmids is required to prevent them from religation.
Hello Florence,
Phosphatase treatment helps no doubt. However, use Shrimp Alkaline Phosphatase instead of CIP, for SAP can be heat inactivated. Alternatively, Fast AP from Thermo also works very well.
Hello Florence,
As I understand, your concern is whether religation might occur in vivo after introducing the linearized plasmids into the yeast. I am not aware of the problem, but don't forget that even if you dephosphorylate your DNA prior to transformation, it may get phosphorylated again in vivo by kinases of the host. This is unlike in vitro ligation, where there is no kinase to rephosphorylate the DNA. Perhaps a good idea would be to try to transform in parallel with the same DNA treated and untreated with phosphatase. If it makes no difference, you will know that it not worth it to perform the phosphatase treatment
The goal of this experiment is to compare the religation efficiency between wt and mutant strains. Cells are grown onto selective media, so they can not grow unless the plasmid is religated in vivo, replicated and passed on to daughter cells. What I'm worried about is self religation of digested plamid in vitro before transformation which can affect the results of my experiment. What I need to know is when people do NHEJ assay using this approach, whether they typically CIP treat or not.
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