Subcloning problems

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Wed Mar 18 11:24:02 EST 1998


In article <01bd5256$bcd3bca0$dc5dfec2 at bio.univ-paris5.fr>, "Cynthia
Marie-Claire" <marie at pharmacie.univ-paris5.fr> wrote:

 
> 2) Among those 70% contain the cloning vector (A) and only 30% the
> expression vector (B). Unfortunately none of these had the insert.

This sounds a bit strange since Nco I and Eco RI are incompatible ends
and, as such, your fully digested cloning vector should not be able to
self-ligate. If it did ligate the assumption would be that the cloning
vector-insert was cut by one of the enzymes and not by the second.
However, your screening indicates that even these clones do not have
insert. This leads me to suspect that your cloning vector may have a
second (cryptic) restriction site for one of the enzymes, and that this
site flanks your insert in such a manner as to generate a cloning vector
fragment but now with compatible ends.

e.g.     -------(Eco RI)----Nco I**********Eco RI-------    -- = vector
                                                            ** = insert
                        Digest with Nco I/Eco RI

 Nco I**********Eco RI  + -------(Eco RI)          Eco RI-------

Recommendations:

1. Verify that your cloning vector only has a single site for each of
these enzymes.
2.  If insert and vector fragments are very similar in size following 
Nco I/Eco RI cutting then choose a unique RE in the vector and digest; the
resulting vector fragments should now be appreciably different in size
from your desired insert to allow vector-free isolation of the insert.
3.  If it is possible, choose a restriction enzyme in the MCS of your
expression vector which would be missing if your desired Nco I/Eco RI got
in but would be present in the expression vector if a religation took
place. This is performed following the ligation step.

An alternative to #3 would be to do your transformation with undigested
but ligated DNA, transfer an aliquot of these cells to a rich broth
culture (superbroth, terrificbroth) with appropriate antibiotics, let this
mixed culture grow for 6-8 hours, extract total plasmid and digest with
the vector-only RE and retransform the digest mix. This is more work but
should highly enrich for expression vector plus insert.

Just some thoughts

Karl the hepB guy

(posted and mailed)



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