Adding linkers to a vector

[Michael Benedik]

In article <2147 at rwja.umdnj.edu>, bawagan at rwja.umdnj.edu (Hinayana Bawagan) writes:
>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.


Did you phosphorylate your linkers??? If you didn't then both your vector and
your linkers don't have a terminal phosphate and you won't get any ligation
products?

If it still doesn't work, then kinase your linkers with some gamma-P32 and
then ligate to your vector. You should be able to run a gel and see some
counts ligated to the vector band after autoradiography. Using labelled linkers
you can troubleshoot your different steps in the procedure.

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 Michael Benedik				INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik at uhou
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