5 replies to this topic

[gulash]

I've doube digested my pCMV5 vector with EcoRI an BglII, and as I've got some problem with it self-ligating, i've dephosphorylated it with CIAP. Now, do I need my ligase buffer to be free of ATP?? ( the insert is still phosphorylated after the same double digestion)

[prabhubct]

you can try ligating it directly.
Why r u asking "Now, do I need my ligase buffer to be free of ATP??"

[gulash]

because as the CIAP is not heat inactivated i have to clean-up the reaction, and resuspend it in a ligase buffer (containg ATP); i'm asking whether the ligase with atp won't rephosphorylase the vector making it capable to self ligate once more

[prabhubct]

T4 polynucleotide kinase need to be present in reaction to make vector rephosphorylated. your ligase reaction do not have  it. so no need to worry abt ATP.

[gulash]

thx very much!!

[phage434]

You should switch to shrimp alkaline phosphatase or antarctic phosphatase, both of which can be heat killed, avoiding a painful cleanup step.  The less you do to your DNA, the fewer opportunities for problems.