Kinasing a probe with T4 polynucleotide kinase

Jim Smith
Thu Oct 5 15:25:16 EST 1995


HI !

I am having trouble making a prode using double-stranded DNA.

I cut my DNA with Pvu II or EcoRV and then phosphatase the
DNA for 1 hour (Promega calf alkaline phosphatase) I then 
phenol extract and ethanol ppte the DNA and then kinase the
5' ends using T4 polynucleotide kinase (promega) and gamma 32P-ATP
for 30 mins at 37 C.
	I then gel isolate the probe...but even when I cut the
band out of the gel (pre-isolation) the gel slab is not
very hot at all...
I am wondering why this is not working !!!!

I have tried to use fresh enzymes and radioactivity and it still does
not work.. I am at a loss.

When I do this procedure with Klenow (on a EcoRI end)..it always works !


If you have any suggestions of comments please post a reply on the
molbionet as my email account is temporarily inaccessible

Thanks very much,
Jim S.



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