Cloning 5kb inserts
sergioal at bbm1.ucm.es
Thu Jan 18 04:33:51 EST 2001
> "Dr. Duncan Clark" wrote:
> > In article <3A655F8E.7598.3B007A at localhost>, the eminent Janakiraman
> > Vasantharajan at K-State Plant Pathology wrote
> > >I had attempted to clone a 5kb insert (from a genomic library) into
> > >the bluescript vector atleast 3 times (into Kpn I - Sma I sites) with
> > >nosuccess. I am doing this only to sequence my inserts. I do a blue
> > >white screening, none of my white colonies contain the insert. One
> > >of my colleagues suggested using the pUC 19 vector.
> > >Any suggestions ??
> > Assuming you are doing everything right then my general conclusion would
> > be that something in that insert is lethal. As you are using a vector
> > with a strong promoter (lac) and screening by blue white you could be
> > simply inducing some gene product that is lethal.
> If you are dealing with a lethal insert, maybe it's a good advice to use
> pUC18/pUC19 (same vector, simetric MCS) so you could try different insert
> orientations, or use the simetric MCS pBluescript (KS or SK).
> I would also suggest to use Xma I (sticky ends) inestead of Sma I (blunt
> ends), which cuts at 37ºC (as Kpn I does), so you could perform the double
> digestion at the same time.
> Hope this helps.
I forgot to say than sometimes the right clon is blue, because the insert
contains a promoter and a ORF in the same reading frame than the lacZ'. Use
the alkaline phosphatase on the vector (to avoid religations) and check some
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