Help!!!Restriction enzyme digestion troubles.

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.

Please reply

i am not getting any colony after ligation & transformation

While getting colony in non contiguously present RE site,using same vector



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On Mon, 26 Jun 2006 08:03:45 +0100 (BST), shyam nandi
<[Only registered users see links. ].in> wrote:


Probably not. Digest with PstI first, then KpnI.

Check NEB catalog for information on how many bases are required for
good digest.

In <[Only registered users see links. ].net >,
shyam nandi <[Only registered users see links. ].in> wrote:


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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