In a previous article, haviland at kids.wustl.edu wrote:
>In a previous article, rrumpf at magnus.acs.ohio-state.edu (Robert Rumpf) wrote:
>>I've heard that the alpha-complementation-based color selection using
>>lacZ-phagemids, lacZ deletion host cells, and Xgal/IPTG plates can also
>>using the same phagemids/host cells but plated onto MacConkey's agar. Is
>>true, how is it done, and what's the mechanism for color selection? I
>>appreciate any help you can give...
>Hi Bob: I'd strongly suggest that you look up
>Jennings and Beacham in Biotechniques vol 7:1082 (1989).
>Once our lab lacthed onto this method we rarely plate our ligations on
>anything else unless dictated by a particular vector that doesn't have the
>Betal-gal. What is critical is that the bug of choice have the lac
>deletion. HB101's don't work, but Sure's, JM109, TOP10F' and others work
>just fine. At the time of plating you just put in 10 ul of 100mM ITPG and
>voila! You have red/white color selection. One thing though, over time
>your white (recombinant) colonies will turn a slight shade of pink making
>colonly selection after about 24-48 hours very difficult. So if you do
>your ligations one day and will need to pick on another (for whatever
>reason), then one must mark which ones to take or not to take.
>>Hope this helps,
>+ David L. Haviland, Ph.D. Internet:"haviland at kids.wustl.edu" +
>+ Washington Univ. School of Med. A.K.A : The Compiler +
>+ Dept. of Peds./Pulm. Box 8116 ICBM-Net : Just hit St. Louis +
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Further to this discussion:
Pendola and Palis have reported that MacConkey-II lactose agar from Becton
Dickinson Microbiology Systems is able to distinguish recombinant plasmids
(Puc-9, pBluescript, pGEM-4) without the use of IPTG (Trends in Genetics,
Vol 9, pg 3, 1993).
Greg Denomme, PhD
denomme at FHS.McMaster.ca