Adding linkers to a vector

Hinayana Bawagan bawagan at rwja.umdnj.edu
Fri May 7 11:33:11 EST 1993


Here's the purpose:  I have a vector in w/c the RE site used to linearize 
it for transcription is found in my insert.  Had trouble doing partial 
digestion.  So I want to remove that site and put in a linker in w/c
the RE site is not found in my vector nor insert.

Here's the procedure:

1.  Linearize the vector at Spe I site.
2.  Fill in w/ Klenow to produce blunt ends.
3.  Dephosphorylate the vector w/ CIAP.
4.  Ligation of deP vector and Pac I linkers.
5.  Digestion w/ Pac I RE to remove the concatemers and
    leave sticky Pac I ends.
6.  Circularization of the modified vector.
7.  Transformation.

At each step, phenol/CHCl3 extraction and Etol precipitation was done.

Can't do it.
Please help.  Any advice will be useful and appreciated.



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