Ligation of fragment with both blunt and cohesive end

David Micklem seemysignature at mole.bio.cam.ac.-uk
Fri Jan 10 08:53:04 EST 1997


In article <32D63C6D.A18 at sheffield.ac.uk>, K.Mulcahy at sheffield.ac.uk wrote:


>1. Do I need to purify the DNA following fill-in/trimming before I cut
>with the second enzyme (BamHI) or will heat-treatment (75°C for 10
>mins) be sufficient to inactivate the fill-in/trimming enzyme so that
>it doesn't blunt the BamHI cut?

Personally I'd phenol/chloroform extract, and ethanol precipitate (with
glycogen or linear acrylamide carrier).  It doesn't take long, and should
definitely work.  It ISN'T necessary to waste hours chilling the
precipitate. Just spin immediately for as long as you can be bothered
waiting (>5 mins).
>
>2. To ligate types of both ends at the same time by do you (a) use the
>amount of ligase that would be used for a routine blunt-end ligation
>where all the ends are blunt, and (b) do the ligation at 14-15°C as
>for cohesive-end ligation?

Um, I always do them exactly the same way anyway, using NEB ligase and
buffer! Incubation at room temperature for 15mins for cohesive ends and
>1hr for blunts should be fine...

Hope it works,

David

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.-uk
Fax: [+44] (0)1223 334089  
** To avoid unsolicited Email I have doctored my return address**
**Please use the above, with the "-" sign edited out.                   **            

_____________________________________________________________



More information about the Methods mailing list