Blunt ending??

[Thibaud Le Mouël]

In article # 59267 Farid wrote :
>
>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.
>
>Any info would be greatly appreciated.
>
>Farid Jalali
>Mushroom Research Group
>University of Toronto at Mississauga
>
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I think it's better to use klenow for both 3' and 5' protruding :

Klenow has a 3'->5' exonuclease activity and a 5'->3' polymerase activity
so this is the method I use :

	  8 microl of DNA in water
	+ 1 microl KGB 10X
	+ 1 microl Klenow Fragment

	5 minutes at 37 degrees (this step is for 3'->5' exonuclease activity)

	+ 2 microl dNTP (0,125 mM each)
	+ 1 microl Klenow fragment

	5 minutes at 37 degrees (this step is for 5'->3' polymerase activity)

Make a phenol/CHCl3 and precipitate it

For more informations see Maniatis pp 5.40 to 5.43

Bye
Thib

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