Cloning 5kb inserts
sergioal at bbm1.ucm.es
Thu Jan 18 04:30:27 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.
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