rennie at bldghsc.lan1.umanitoba.ca
Tue Sep 26 17:45:24 EST 1995
I'm amazed at all the troubles people seem to have doing blunt ligations.
In the lab I'm in we do them routinely. I even blunt ligated a 500 bp PCR
fragment first try for a friend of mine. I think discussion needs to be
kept up about this area because obviously everyone hasn't read the
literature/worked out all the bugs.
I don't want to write an essay here but I'll try to summarize all the
info I've been told about doing these ligations
1) pmoles of ends needs to be equal to 3:1 insert:vector
- we estimate off an agarose gel using EtBr band intensity keeping in mind
2) our Nick translation buffer is a little different than published in Maniatis
and I may even convince my boss to publish this...
3) Yes ppt your ligation its amazing the difference this makes. Use NaOAc
EtOH to get as much down as possible.
Sorry, gotta go good luck to all
we're not happy if our yeast ain't blue!
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