strange cloning result
zzalforr at fox.uq.net.au
Tue Mar 28 20:50:50 EST 2000
Yeah seems to be variable. I get both situations quite often.
When I get fewer on the ligation plate versus the control ligation I've
assumed there is something inhibiting the formation of circular
clones. Either because the insert is in excess and there is heaps of
linear concatemers OR that there's something inhibitory coming through in
the insert prep thats inhibiting the ligase.
However most of the time the colonies I do get do contain the
insert (thankfully :).
One thing that I do routinely is if the cloning sites in the vector are
close together (ie. 10-20 bases), dephosphorylate. As you can't be sure
when cutting a band from a gel that it is single or double cut.
This will cut the "background" to close to zero and give you the ratios
you were after :).
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