cloning concatamers

Slawek Slawek
Mon Jan 16 21:16:01 EST 1995


In article <3fea5k$20j at mark.ucdavis.edu> szcooley at dale.ucdavis.edu (Michael Cooley) writes:
>Dr. S. Bhattacharya (sbhattac at hgmp.mrc.ac.uk) wrote:
>: Can anyone guide me to a reference describing how to clone
>: concatemers of an operator site without getting inversions,
>: i.e. all units of the concatemer in the same direction. I
>: need to clone them upstream of a reporter gene for transcription
>: assays. Many thanks
>: Shoumo
>
>: S Bhattacharya
>: Dana-Farber Cancer Inst.
>: Boston MA

Try following:
1. Clone desired fragment between BamHI and BglII site of a
polylinker.  Sorry, I can't remember a vector with both sites.
Alterantively another pair of six-cutters with compatible sticky 
ends could be used (XbaI/SpeI, XhoI/SalI).
2. Digest resulting plasmid with both enzymes.
3. Isolate the fragment with your favorable method.  Ligate
in a very high concentration of DNA.  Digest with both enzymes.
You can ligate the mixture straight to your vector or isolate a 
concatamer of the desired size and clone it.  Hope it helps.
Cheers.

Slawek.
slawek at rsbs-central.anu.edu.au



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