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pcDNA 3.1/Myc-His(+) B cutting problems

pcDNA 3.1/Myc-His(+) B cutting problems - Protocols and Methods Forum

pcDNA 3.1/Myc-His(+) B cutting problems - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 01-29-2008, 03:07 AM
yitingw@gmail.com
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Default pcDNA 3.1/Myc-His(+) B cutting problems



I have some problem by using pcDNA 3.1/Myc-His(+) B.

I am trying to cut Hind3 and Kpn1cutting sites in order to insert my
favorite gene.

Since I have tried several times and still can not get my favorite
gene inserted, I was wondering

is it possible to cut pcDNA 3.1/Myc-His(+)B by Hind3 and Kpn1 at the
same time?

Would the two restriction enzymes compete with each other since their
cutting sits are near by?

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Old 01-29-2008, 04:52 AM
DK
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Default pcDNA 3.1/Myc-His(+) B cutting problems

In article <[Only registered users see links. ]>, [Only registered users see links. ] wrote:

Their sites are right next to each other, which might indeed be
a problem. It's not competition but the fact that many restriction
enzymes need extra dsDNA on both ends of their recognition sequence.

KpnI is is supposed to cut well (50-100% activity) with just
one base beyond recognition site. HindIII, however, is more
demanding: no cut at all with one base beyond its recognition,
0-20% with two bases, 50-100% for three (according to Fermentas
table).

So, apart from using different enzyme pair or doing restriction
enzyme-independent cloning, your choices are:
1) Do HindIII digestion in NEB buffer 2, then, after verifying that
it is complete, throw in as much KpnI as reaction conditions afford
and hope for the best.
2) Clone something/anything into HindIII site (obviously, orientation
does not matter in this case; any fragment or 20 bp annealed oligos),
then the double cut will be efficient. (And, in the case of large fragment
inserted, double cut will be eay to monitor/verify).

DK
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