I have a vector with PfoI site where I am planning to insert my gene into that site. However, PfoI is only available at thermo and I use this enzyme over the vector using tango buffer. I might need to dephoshporylate 5' end using CIP, but I am not sure weather CIP would be active in tango buffer. If anybody has experienced this kind of problem, please help.
Cloning is a very tedious task, and I would suggest that you digest your vector, purify it by the classical Phenol-chloroform method and then CIP treat it and then purify it again. After that, perform a self ligation assay, and gel elute the linear unligated product from LMA by the classical method, without a kit. If you start with almost 10ug of vector DNA, you are sure to retrieve sufficient. Best of luck !!!
Dear Venkat,
Tango buffer is generally used for DpnI enzyme. But if you want to use it for CIP do the following:
Check the composition of tango buffer.
Check the components necessary for CIP.
If the two match, go ahead.
Best,
Jogender
Not sure about CIP, but since you're already with Thermo, you may consider using their Fast Alkaline Phosphatase, which is compatible with Thermo's restriction buffers (simultaneous digestion and dephosphorylation possible). Saves time and is not particularly expensive (just about like a further restriction enzyme).
I do not have any interests in Thermo, this is a suggestion, not advertising ;-)
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