blunt end !!!

edd at okstate.edu edd at okstate.edu
Mon Dec 23 16:18:00 EST 1996


On 20 Dec 1996, =A7=DA=ACO=B7R=B7=D3=C3=E8=A4l=AA=BAmutant! wrote:

> Help!Help!Help!
> I have tried blunt end ligation for two mouths.
> But the cloning was still not done.
> Does anybody have good method?
> Please help me!
> Thanks.
>                         isabella Chen
>=20
>=20
Isabella,
=09I've found that there are two approaches that work for me. =20
First, use a lot of vector and insert DNA (100 to 500 ng each) in the=20
ligation reaction, be sure to use fresh ligase and 1X buffer and ligate=20
o/n or longer at 15 C.  Then transform cells (I use CaCl2 treated=20
competent cells) with 2, 4, and 8 microliters of the ligation reaction. =20
If you have super-competent cells that's better.  Second approach, (if an=
=20
option) is to use the 3' non-template dependent activity of taq=20
polymerase to add a single 3' A to the insert (or vector) and a single T
to the vector (or insert).  Although the second approach is an avoidance=20
approach to blunt-ended cloning it does work well.
=09Good luck!!!
=09=09Ed




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