How much ampicillin?
chrisb at hgu.mrc.ac.uk
Wed Sep 25 09:45:53 EST 1996
Bill Burnett (bbur at wpo.nerc.ac.uk) wrote:
: Promega recommend a final concentration of ampicillin in
: LB/ampicillin/IPTG/X-gal plates (for E. coli JM 109 transformed with
: T-vector) of 100 ug/ml... Can I get away with only 50 ug/ml as
: suggested by a lab manual or is this a false economy?
Yes, you can get away with it, and millions do. But you DO get more
satellite colonies, which can make the plates rather messy,
particularly at high plating densities. Ampicillin is cheap compared
to X-gal and IPTG, after all.
: Anyone got a sure fire cheap plate recipe?
Unfortunately, JM109 is laci^q, so you probably need a bit of IPTG to
fully derepress the lac promoter. You could try a test titration
downwards from the recommended level of IPTG to see how little you can
get away with (i.e., which still allows blue/white discrimination). You
can also reduce the X-gal quite a bit, but blues take longer to show up
if you do. Alternatively, if you don't need the phagemid properties of
the T vector, you could use a non-laci^q host such as JM83 or DH10B
instead of JM109 and leave out the IPTG altogether.
Of course, if Promega's T-tailed vector prep is of sufficiently high
quality, it should have negligible amounts of uncut or untailed
plasmid, so in principle all the colonies you get should have inserts,
and you can omit X-gal and IPTG. (Don't try this at home, kids.)
Chris Boyd | from, | MRC Human Genetics Unit
chrisb at hgu.mrc.ac.uk | not | Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb | for | Edinburgh EH4 2XU, SCOTLAND
More information about the Methods