Help!!!Restriction enzyme digestion troubles.

Aawara Chowdhury aawara at FEMA-trailer.org
Wed Jun 28 05:36:46 EST 2006


In <mailman.90.1151341317.23079.methods at net.bio.net>,
 shyam nandi <biotech_iicb at yahoo.co.in> wrote:

> i am doing  double digest restriction digestion of baculogold vector with
>   RE pstI &  kpnI
>   the both sites are contigously present one after another in the vector
>   liki 
>                             .....CTGCAGGGTACC......      (vector)
>                                  pstI        I       kpnI
>    
>   Is my digestion should complete successfully.
>    

Your sites are likely too close to each other.  Either use different
sites, or do a three-way ligation.  We've done three-way ligations in
a similar situation several times over the last year, and it has been
successful every time.

For this, first identify another unique restriction site that is in
your BaculoGold vector, and is at least 1 - 2 kb removed from the
PstI and KpnI sites.  Let's call the enzyme that cuts here as "XYZ".

a) Cut with XYZ + PstI; isolate the large fragment
b) Cut with XYZ + KpnI; isolate the small fragment
c) Do a ligation with your PstI/KpnI-digested insert, the large 
   fragment from "a", and the small fragment from "b".

Something else that you should keep in mind is that both PstI and
KpnI generate 3'-overhangs.  We find these ligate much more efficiently
if we drop the ATP concentration in the ligation from 1 mM to 0.2 mM.

AC
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