4 fragment ligations.....?
jpcd100 at mole.bio.cam.ac.uk
Tue Aug 22 10:00:35 EST 2000
In article <mantei-865020.12254822082000 at news.ethz.ch>, Ned Mantei
<mantei at cell.biol.ethz.ch> wrote:
> 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.
How important is the molar ratio of the various frags, Ned?
Because I hate having to fiddle about with many vector to insert
ratios, I mainly try to do two way ligations which work fine whatever
(more or less) but I've had poor to middling results with >3 frags -
would that chain reaction cloning procedure you cited get round
problems of low or incorrect concentrations of some fragments do you
think? (I have read the ref, but I'll need to read it again a few times
to get my head round it).
John Dixon Lab 44 (1223) 334131
Wellcome/CRC Institute Fax 44 (1223) 334089
United Kingdom CB2 1QR e-m: jpcd100 at mole.bio.cam.ac.uk
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