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vector dephosphorylation

cloning dephosphorylation

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5 replies to this topic

#1 gulash

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Posted 11 October 2012 - 08:29 AM

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)

#2 Inbox

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Posted 11 October 2012 - 10:06 AM

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

#3 gulash

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Posted 11 October 2012 - 10:29 AM

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

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Posted 11 October 2012 - 10:40 AM

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.

#5 gulash

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Posted 11 October 2012 - 10:47 AM

thx very much!!

#6 phage434

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Posted 11 October 2012 - 02:15 PM

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.





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