I have digested my vector and PCR product with restriction enzymes A and B (both blunt end cutters).
For ligation I have either the vector or vector + insert. Now as it is a blunt end digestion, both of these should give me colony after transformation (selection marker kanamycin).
What should I do (other than re-digestion after transformation) to to get rid of colony in negative control after blunt end digestion followed by ligation?
Also how to increase probability of vector + insert ligation rather than vector ligation.
Parijat
I don't typically use negative controls when doing blunt-end cloning, but I can tell you that if you flood your reaction with at least 3 times as much insert as plasmid (by molarity) the insert will get ligated into your plasmid with enough efficiency to be able to obtain your target product.
I honestly add as much insert as I can to this (i.e. I don't dilute it and use the maximum amount possible for the volume I am working with). I have never encountered an issue of having too much insert...
Negative control of what ? transformation or ligation?
if your getting colonies in transfomration negative control then your competent cell might have contaminated with circulariezed plasmid dna or cross contamination of cells. If it is ligation the same would be the chance (without ligase).
However for increasing ligation efficiency you can try ligation in presence of PEG, with different ratio of insert : vector (1:1, 1:3 and 1:6) .
You should dephosphorylate (calf intestinal phosphatase or shrimp alkaline phosphatase) the blunt ended vector after digestion prior to setting up the ligation to prevent the vector from ligating back to itself and reduce the number of background colonies in you vector plus ligase (no insert) control plates.
Dear all,
Thank you for your valuable comments !
I want to do a negative control to know minimum how many colonies I have to screen in the (vector+gene) ligation plate.
With regards,
Parijat
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