I'm trying to clone my insert in pet 16b with nde1 and xho1 restrictions enzymes but I'm always getting only self ligated vector ,and the vector control also have many colonies than the insert-vector ligated.so I decided to do the vector digestion first with one enzyme and after purification, make it with the second enzyme. I'm wondering if it is also possible to do the same with the insert?
It might be by the problem with cleavage close to the end of DNA fragments.
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
https://www.neb.com/-/media/nebus/files/chart-image/cleavage_olignucleotides_old.pdf?la=en&rev=c2f94e1cdcd549c5bf8fdb59f7b63f67&hash=4FBF94A3878D7C5E79BC1DBBADD5CCDF70803D06
I hope it helps you.
the base pairs are 5 from each end, what is surprising is that the vector control is giving many colonies, but I've done digestion with the 2 enzymes during 4h for both insert and vector
Abire Sigbessia What about 2nd enzyme activity?If you've already checked both of 2 enzyme (NdeI, XhoI) activities, purified DNA fragments after 1st digestion may have some problems, remained ethanol, etc. Good luck.
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