Cloning 5kb inserts
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Wed Jan 17 10:52:05 EST 2001
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.
So try plating your cells in the presence of glucose and no IPTG. Re-
grid umpteen on plates with and without IPTG/XGAl and see if you pick up
colonies that will not grow in the presence of IPTG i.e. lethal.
If that is no go then think about using a lower copy vector i.e. pBR322
and/or a vector with no strong promoter flanking your insert.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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