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activity of T4 polynucleotide kinase - (Jun/22/2005 )

I wish to use the T4 DNA kinase to phosphorelate the double strand DNA before I proceed to the next step of cloning, ligation. And I have a few questions to ask. First, does DNA kinase phosphorelate the double strand DNA that is a blunt end DNA with 5' OH? Second, If I have 3' overhang double strand DNA, can kinase phosphorelate this polynucleotide?

Thank you for your help

-Ekkaluck_k-

Hi,

The answer is yes T4 Polynucleotide Kinase can phosphorylate blunt and 3' overhang double stranded DNA, however it is not as efficient. NEB recommends heating to 70C for 5 minutes and chilling on ice prior to kinase addition and/or addition of PEG-8000 to 5%.

Hope this helps

Scott

-Scott-