Help needed for cloning problem

ChenHA hachen at bc.ic.ac.uk
Sun Aug 15 10:05:50 EST 1999


On 11 Aug 1999, Alexandre Mooser wrote:

> A very stupid problem for a standardized procedure.....:-(((
> I try to ligate several PCR products (400-500 bp)  into an expression
> vector (PQE-derived). I digest (2 different RE, sticky ends) AND
> dephosphorylate the vector. After purification, I ligate 30-50 ng vector
> with several concentrations of digested inserts (ratio 1:3, 1:5), 3hrs @
> RT. I load the ligations on a gel, and get a band which corresponds to a
> vector dimer or even trimer (according to length) ! All the controls are
> ok: no ligase with insert, no insert but with ligase, vector alone
> without insert and ligase.
> 
> I nevertheless transform competent cells, repick a few clones to screen
> for the insert, and vector and insert are present (both at correct
> size), but the undigested construct (vector + insert) still gives a much
> too higher band (dimer or trimer...) !!!
> Expression (IPTG induced) gives nothing.
> 
> PLEASE, HELP ME.....!!
> 

I'm not sure which bit you are worry about, the absence of protein
expression or the presence of religated vectors.  As long as you get
what you want (i.e. insert in vector), there is no need to worry about
religated vectors.  It is obvious that the phosphatase treatment
hasn't worked too well, however, I don't think this is something you
need to worry about as you don't need to dephosphrylate your DNA when
you do directional cloning (I never do and I would recommend that you
don't).  I would suspect that the problem is with DNA digest, either
of the vector or your PCR product, more likely the latter. If you
haven't added enough extra bases to the ends, you might find that the
RE might not work too well.  Therefore you should either make new
oligos, or clone your PCR product into a TA-vector (or blunt-ended
cloning) and then sub-clone into your expression vector.   When you do
the cloning into your expression vector, make sure that the vector is
well digested and also do another ligation at 5X dilution of DNA
concentration.

As for protein expression, you should check first by sequencing that
your insert is ligated in frame (if there is a problem with the RE
digest your insert may not be correctly ligated).  If everything is OK
and there is still no expression, then this is a different problem all
together and there are too many possible reasons to go into right now.
Feel free to e-mail me if this is the case.
 





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