Insert alone gives colonies?
no.user at anon.xg.nu
Sun Aug 20 11:06:16 EST 2000
Anybody ever seen something like this before? On a
ligation of a long PCR fragment, much troubleshooting
has shown that colonies come up on an insert alone +
ligase control plate. (Minipreps on colonies from the
vector + insert plate are either vector without insert
(rarely) or (mostly) some small garbage plasmid,
smaller than the starting vector. Also, a vector +
insert without ligase control plate gives few or no
colonies, suggesting that the colonies on the insert +
ligase plate arise through ligation.)
The two possibilities I can think of are: 1)
contamination of the insert with a rogue (linear)
plasmid (somehow-- but everything is autoclaved,
gel-isolated, not exposed to UV, treated carefully,
etc.) or 2) somehow the insert itself is generating
the plasmid (though you'd have to argue that somehow
it's getting an amp resistance gene, an origin of
replication, etc.-- seems pretty unlikely). Any
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