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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