cloning problem...

Arnoud van Vliet avvliet at knoware.nl
Mon Jul 31 15:42:28 EST 2000


> We have been trying to clone a human gene in to TA vector. We've got
> blue/white selection,  have screened the white ones directly by PCR, and
> then tried to grow up those with inserts - this is where the problem
> starts. On trying to grow up for mini-preps, nothing happens. There is
> absolutley no growth.
> We have tried different media and both Kan and Amp selections (as well
> as no antibiotics at all), but we just cannot get any of these colonies
> to grow in liquid media - except for once when it turned out that the
> insert had two base pair changes.

It looks like the insert is toxic for the E. coli. The PCR works as there
might be one or two dormant cells with the plasmids.

Try growing at 30 degrees C, with lots of glucose in the medium (to repress
any activity from the lac promoter). Alternatively, try cloning in a low
copy number vector. There are vectors called pWSK.. (don't remember the
number), which have a very low copy number ori, and a lacZ gene for the
blue-white screening.

Do you need the whole sequence, or can you maybe clone it in multiple
smaller parts?

hope this helps
Arnoud







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