three fragment cloning

Frank Maiwald maiwald at mpiz-koeln.mpg.de
Thu Oct 1 09:50:01 EST 1998


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



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