RE digest and klenow - sequential blunting (Oct/26/2006 )
just a liitle question...
Is it possible to use klenow enzyme directly in a restriction digestion reaction?
e.g. Im digesting in 50ul total volume for 2h with BamHI (NEB buffer 2, blue + BSA), now can I just add 1um of 10mM dNTPs and Klenow and put the whole reaction on 25°C?
Will that work for filling up (blunting) the BamHi digested sites (5'- overhangs) ?
thanks & have a nice day!
It depends on the Klenow you use. Some datasheets suggest you use a Klenow buffer otherwise your enzyme won't work. Others say you can fill in in the digestion buffer. Anyway, I would heat-inactivate BamH1 first if you want to add dNTPs and Klenow to the digestion.
thanks for replay
BamHI cannot be heat inactivated
Im using Klenow from Roche
NEB buffer 2 is the "official" klenow buffer that NEB suggest on their webpage
bam h1 seems not able to be heat inactivated.
So a phenol chlo should be good.
If not possible, considering the fact that BamHI is not really active at 25° and the fact the klenow would not restore the whole BamHI site, should be feasible.
But if you need strictly blunt ends, a pcr with no-overhang-adding enyme should be easy and will produce blunt ends too.
To be on the safe side I would do a phenol/chlorophorm. Anyway you could give it a try because BamH1 activity shouldn't be so high anymore after 2 hours at 37 and it shouldn't be active at 25 either
well in my opinion, it does not matter how active Bam still is, since Klenow will not restore the BamHI site..?