blunt ligation problem

S. Simon renniesl at ?cc.umanitoba.ca?
Wed Aug 19 16:09:08 EST 1998


In article <1998Aug17.155745.6440 at leeds.ac.uk>, medbh at biovax.leeds.ac.uk wrote:

> In answer to queries:  1.  To generate a blunt ended plasmid, I cut it
with SmaI.  2.  I did also dephosphatase the plasmid.  This worked as I
tested this by comparing normal digested plasmid to dephosphatased plasmid
when transforming the bacteria.

Hi we blunt PCR products in that range into a SmaI cut pUC vector on a
regular basis...so this will work : )
We typically treat the PCR product with Klenow to "polish" the ends and
then Kinase to add a P'se group
Use ~200 ng vector and 3 -5x insert conc. in a 25 ul rxn.   (and just to
plug my friends company) ...we swear by Ligase and Cip from
Boehringer-Mannheim .  Use 5 ul of Ligase .....and that's about it.

Hope this helps some.
Sharon
you can contact me at :    renniesl at cc.umanitoba.ca

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