4 fragment ligations.....?
mantei at cell.biol.ethz.ch
Tue Aug 22 05:25:48 EST 2000
In article <39A14326.D7814A9F at biocomp.unl.edu>, Chris LaRosa
<clarosa at biocomp.unl.edu> wrote:
>Recently there was a discussion on the feasiblility of complex
>I am contemplating attempting a 4 dna fragment ligation. The constuct
>will be directional with all sticky ends. Any comments or references
>would be appreciated.
I have done ligations with as many as 5 fragments (vector, 3 fragments,
double-stranded olio). The conditions you mention (directional, all
sticky ends) very much favor success.
Things I considered in my own constructs:
1) All pairs of ends were different, so that the fragments could only
fit together in one way.
2) There was only one pair of blunt ends. I would not attempt a many-way
ligation with more than that, as the efficiency is too low.
3) For a 5-way ligation, the frequency of correct clones is rather low.
In some cases I did a colony lift and hybridization to find the few % of
colonies with the main insert fragment. This shouldn't be necessary for
a 4-way ligation.
4) There can be problems with contamination from vectors from which the
various insert fragments were isolated. When isolating insert fragments,
I often cut with additional enzymes so that the (contaminating) vector
5) Even if the final vector had two different ends, I treated it with
Finally, if I had such projects to do again, I would consider the method
described by Pachuk, et al., Gene 243:19-25, 2000, "Chain reaction
cloning: a one-step method for directional ligation of multiple DNA
fragments". The method seems very clever, but I haven't tried it myself.
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
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