epatters at uwo.ca
Sun Nov 10 22:37:53 EST 2002
If you're using just a a run of the mill plasmid, my first guess is
you're REALLY not supposed to try to stick a 14kb insert into a 4kb
plasmid. From what I recall plasmids generally can only handle <7kb.
Read the plasmids manual and it will probably say in the intro what the
maximum size is. You'll probably need a cosmid for something that's
14kb. I've not ever worked with them before though because I've never
stuck more than 2kb into a plasmid.
dygibbs at hotmail.com wrote:
> I have attempted to clone a 14 kb insert (Mlu I/Xho I) into a 4 kb vector (Mlu I/Xho I). The vector has been gel purified and dephosphorylated (shrimp alkaline phosphotase), the fragments ligated (NEB T4 ligase overnight at 16 C), and transformed into XL-1 supercompetent cells (Stratagene). After plating/overnight incubation, very few colonies are present, and after screening for positives with Mlu I/Xho I, there is no insert. Any suggestion/tips?
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