background colonies

Sergio Alonso sergioal at
Wed Oct 3 05:36:55 EST 2001

Which microorganism, strain, antibiotic, transformation method?
Check the plates before plating. If they are too old the antibiotic
could be degraded.
Don't plate too many cfu per plate if the selection antibiotic is
ampicillin, it gets degraded by the b-lactamase and many non-transformed
cells can grow (satellite colonies)
Ampicillin (an many others) can be added at higher concentrations than
suggested by literature, if you use medium-high copy number plasmids.
For example, E.coli DH5alpha or CC118 with pBluescript grow perfectly at
150 ug/ml of ampicillin, when 50-100 is suggested. I did'nt test it, but
i think they could grow even at higher concentrations.
If you are plating the result of a transformation assay, check the
experiment plating a competent cells vial transformed without any
plasmid, just to verify that the cells are not getting spontaneous
immunity to the antibiotic (something frequent with nalidixic acid and
rifampicin) or they are contaminated with a resistant bug or another

Hope this helps.

Baoshan ha escrito:

> i have a lot of background colonies can you help me get rid of them

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