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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



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