Help with ET recombination systems
Caroline
Does anyone have any experience with using ET
recombination systems?
I have been trying to insert an antibiotic selection
marker into a bacterial artficial chromosome (BAC)
in E.coli by homologous recombination. The selection
marker is part of a linear PCR product, flanked by
60n.t. homology arms. The PCR product is treated by
DpnI digest to remove template DNA and then is
electroporated into E.coli containing the BAC and
recombination plasmid expressing RecE and RecT
proteins. I have tried ampicillin and hygromycin as
selectable markers. I do get resistant colonies
which I screen by PCR, but none of them are
targetted correctly.
I hope someone can give some advice.
Caroline (PhD student)
=====
Moderated
bionet.genome.gene-structure
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Lauren Amable
I too am using ET recombination to modify our BAC and I am
experiencing problems. It sounds like you are doing everything like
you are supposed to. What plasmid are you using for your recombination
and what are the host cells for your bacmid. This could be a problem.
If you are using DH10b cells which are recBC positive than you need to
the gam protein also turned on in order to inhibit these recombinases
b/c they will screw up your speific recombination. Also make sure your
DNA concentrations are good, I had problems with that at first and
realized the purification method I was using was losing my DNA. I am
actually trying to recombine at 2 places on my bacmid and so far i can
only get one to work, I have yet to figure out why the other is not
working. If I may, I have a question for you-what do you electroporate
at? What size cuvette do you use? I am just curious b/c I think I
might be frying my cells, who knows, just wanted some input from
another fellow gradute student. If you have any questions just let me
know!
Sincerely,
Lauren
(another PhD student) :)
c.s.ga...@reading.ac.uk ("Caroline wrote in message news:<aovi9q$cr4$1...@mercury.hgmp.mrc.ac.uk>...