Blunt ending??

Dom Spinella dspinella at chugaibio.com
Mon Jun 8 12:30:23 EST 1998


> I need to blunt end a linearized plasmid with a 5'
> overhang of 2 bases, as well as a fragment of DNA with a 5' overhang.
> The strategy I'm considering at the moment is digestion with Mung-bean
> nuclease. Any thoughts?
> 
> Also, assuming this strategy is okay, will I need to kinase, so that I can
> ligate afterwards.


I would use Klenow + dNTPs to fill in the 5' overhangs rather than Mung
bean nuclease which can sometimes be pretty aggessive and nick up your
DNA.  Regardless, the 5' ends of your DNA will still be phosphorylated
(assuming they started out that way) so kinasing would be superfluous. 
On the contrary, you may wish the treat your plasmid DNA with alkaline
phosphatase and remove the 5' phosphate in order to prevent it from
re-circularizing during your ligation reaction. BTW, this is extremely
elementary stuff, and you really should consider reading through the
relevant sections of Maniatis or the Red Book or some other source of
general cloning information if you're going to fool with this sort of
thing with any regularity. Good luck.
--Dom Spinella



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