Ligating small insert
Gary Kucera
kucera~gt at glaxo.com
Wed Aug 21 07:30:18 EST 1996
"Minh Q. Nguyen" <mqn1 at ea.oac.uci.edu> wrote:
>Hi,
>
>Has anyone tried ligating a very small insert (~50 bp) to your typical
>vector (e.g. Bluescript)? If so, what is the optimal ratio of insert
>to vector and how efficient is the ligation? Any suggestion would be
>appreciated. Thanks.
>
>mqn
I have successfully cloned a 70 bp fragment into pBluescript. The
important factor to consider here is the ratio of ligatable ends. I
used a 1:5 ratio since it was a blunt end ligation.
First determine the number of pMole ends for both insert and vector
using the following equation:
(2 x 10e6)/(#bp x 660) = pMole ends/ug DNA
Then use a 1:5 ratio (vector:insert) of pMole ends in your ligation
reaction.
Happy Cloning.
________________________________________________________
Gary T. Kucera, Scientist
Cellular Sciences/Transgenics Group
GlaxoWellcome Research, Inc.
Five Moore Drive
Research Triangle Park, NC 27709
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