three fragment cloning

Hiranya Roychowdhury hroychow at NMSU.EDU
Fri Oct 2 15:25:35 EST 1998


At 03:50 PM 10/1/98 +0100, Frank Maiwald wrote:
>Dear all,
>
>In a cloning project I am fool enough to try a three fragment cloning.
>My fragments are ca. 3000 bp (vector), 840 bp (insert 1) and 280 bp
>(insert 2). The vector will be cut with ApaI and NotI, insert 1 with
>ApaI and HinfI, and insert 2 with HinfI and NotI.
>
>Does anyone have (positive) experience with three fragment cloning and
>could give advice e.g. on fragment ratios, addition of PEG, low or high
>ATP concentration, ligation conditions?
>
>Any hints are wellcome.
>
>Thanks in advance,
>
>Frank
>
>

This works fine with roughly equimolar amounts of the vector and the
inserts. The only poducts you are going to recover from this lig/trans. is
the product you need AND vector-vector lig. products. So, contrary to one
posted suggestion, dephosphorylation will obviously reduce (if not
eliminate) the occurence of the latter product. Result is the likelyhood of
utilization of all the competent cells by the vector+inserts species.
Dephosphorylation of the vector insures that 90-100% of the picks from the
transformation will carry the insert ( vector religation does occur despite
dephosphorylation, but at a very very low rate).


Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




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