Adding linkers to a vector

Basavaraju Shankarappa bsh at MED.PITT.EDU
Fri May 7 16:14:41 EST 1993


If the insert is of decently smaller size, why not synthesize
two primers from the begining of the transcription start site
to the point where you want it terminated, PCR the insert
and clean it up and use it for transcription.
May be it is not as simple as this, anyway my thoughts.
Raj Shankarappa
bsh at med.pitt.edu
> ------------------------------------------------------------------------------
> 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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