3 replies to this topic

[shimshady]

If i am doing a double digestion, is the calf alkaline phosphatase required?
Thanks

[perneseblue]

shimshady, on Mar 5 2009, 06:13 AM, said:

If i am doing a double digestion, is the calf alkaline phosphatase required?
Thanks

Technically, if the vector is double digested, CIP is not required. It can be skipped and some people do.

Personally I always dephosphorylate my vector. It is an extra safe guard should the digest of the vector fail to go to completion.

However one must be careful when using CIP as over dephosphorylation will damage the end of the vector, preventing ligation.

[shimshady]

So i also have to digest my PCR amplification insert then ligate into my vector (pET-15b).  Should i use CIP on both products?

[little mouse]

no, you only dephosphorylate the vector, not the insert. dephosphorylated ends cannot ligate together. dephosphorylation of the vector is done to prevent self ligation of empty vector. If you dephosphorylate the insert too, you won't be able to ligate it into the vector.