Rules of thumb for messing with vector's LacZ' coding seq?

Clive Tregaskes Clive.tregaskes at bbsrc.ac.uk
Fri Dec 6 13:58:38 EST 1996


prigge at darkwing.uoregon.edu (Mike Prigge) wrote:

>I would like to add a pair of SfiI sites to a vector containing the alpha
>complementation peptide. I could drop in a 27 bp oligo, ie, a 9 aa
>insertion. Should this, most likely, be okay? (I am guessing no since
>cloning small PCR products have given me blue colonies before.) Are there
>any particular amino acids that should be avoided within this fragment?
>Thanks
>Mike
>Michael J. Prigge                     prigge at darkwing.uoregon.edu
>University of Oregon               Institute of Molecular Biology
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I tried replacing the entire bluescript MCS with an artificial one
using two different oligo sets, one of which worked and one didn't.
The one that didn't work encoded mostly hydrophobic aa's so we guessed
that this was somehow affecting the protein structure (assuming the
MCS is stuck on the end of the protein and not actually doing
anything.)  This was a much bigger change than you're suggesting but
might be worth bearing in mind.
Hope this helps
Clive Tregaskes
Institute for Animal Health
Compton, UK
clive.tregaskes at bbsrc.ac.uk





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