Adding linkers to a vector
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.
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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