Cloning 5kb inserts

David Micklem dmicklem at cmgm.nospam.invalid
Wed Jan 17 12:30:17 EST 2001


In article <p.r.ashby-3CA4A0.15532817012001 at dux.dundee.ac.uk>, Peter
Ashby <p.r.ashby at dundee.MAPS.ac.uk> wrote:

>In article <3A655F8E.7598.3B007A at localhost>, vasant at PLANTPATH.KSU.EDU 
>("Janakiraman Vasantharajan") wrote:
>
>> Hi netters,
>> 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 ?? 
>
>You could try Asp718 instead of KpnI.
>
>Peter

And/or XmaI instead of SmaI, so that you get a sticky end instead of
blunt... and it cuts at 37C too. According to the new NEB catalogue,
XmaI isn't even as expensive as it used to be.

You might look at a few blue colonies too... blue/white isn't 100%
reliable.

Other than that, try and work out why it isn't working - what do your
controls look like? Particularly, how many colonies are you getting on
control (ligated vector only) vs vector-with-insert plates?

HTH,

David

-- 
D.R. Micklem,              Time flies like an arrow... 
Dept. of Anatomy,          Fruit flies like a banana.
Cambridge University,      Email:dmicklem at cmgm.stanford.edu 
Cambridge, UK              Phone: +44 (1223) 333776
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