I have Ultraid CFTR plasmid which I should remove the CFTR and get my backbone plasmid. I use AsCI and NotI restriction enzyme and make a blunt end with mungbean neuclease but there isn't any colonies after ligation and transformation to DH5a. Is any suggestions?
The absence of colonies after transformation can have several reasons, in addition to the ligation problem.
Have you tested that your bacterial cells are competent and able to accept a plasmid.
After heat shock or electropration, are your cells still alive? Try to test it on a medium that does not contain any selection pressure.
Also make sure of the concentration of the selection molecule (especially if the selection molecule allows to eliminate empty host cells, like antibiotics).
Hi,
After performing digestion, do you find both of expected fragments in dna gel electrophoresis?
Please also keep in mind that AscI might be inhibited by some of typical salts, including NaCl and NH4OAc.
Do you perform phosphorylation of dna after mnase treatment? Do you find any open circular form of dna after post-mnase ligation?
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