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My white colonies turned blue in frig! Why? - (Sep/24/2010 )

I've stored my plates in frig for a week and yesterday i wanted to select some white colony for colony pcr but i found that almost all of them turned blue and the rest were pale blue! What happened? Does it lost the insert?

-hianghao-

hianghao on Sat Sep 25 00:12:38 2010 said:


I've stored my plates in frig for a week and yesterday i wanted to select some white colony for colony pcr but i found that almost all of them turned blue and the rest were pale blue! What happened? Does it lost the insert?


Nope, it just takes a little while for the concentration of blue pigment to build up. For X-gal Blue-white colour test I usually place my plate of colonies at 4 Celsius overnight.

All blue colonies, including the pale blue ones contain empty vector. Hold the plate up to either a white sheet of paper or a black surface to help tell the difference between pale blue and white.

-perneseblue-

perneseblue on Sat Sep 25 01:06:43 2010 said:


hianghao on Sat Sep 25 00:12:38 2010 said:


I've stored my plates in frig for a week and yesterday i wanted to select some white colony for colony pcr but i found that almost all of them turned blue and the rest were pale blue! What happened? Does it lost the insert?


Nope, it just takes a little while for the concentration of blue pigment to build up. For X-gal Blue-white colour test I usually place my plate of colonies at 4 Celsius overnight.

All blue colonies, including the pale blue ones contain empty vector. Hold the plate up to either a white sheet of paper or a black surface to help tell the difference between pale blue and white.


If it is so, how long should i keep my plate after spreading to ensure the white colonies are the real white colonies? I incubated for 16-20 hours at 37C and picked the white colonies. Only after a week the white colonies turned blue.

-hianghao-

It depends on how much X-gal and IPTG was added to the plate, and what your definition of blue is. Pale blue (almost white) or deep blue.

For me, after spreading the cell culture, I leave it at 37 C for colonies to develop. After that I take the plate and leave it for another 24hr at 4-10 C. That is usually enough to tell the difference between white and pale blue.

-perneseblue-

Sigma sells S-Gal, which makes differentiation of lacZ+ strains far easier. It turns those colonies black, rather than blue, with little possibility of ambiguity.

-phage434-

perneseblue on Sun Sep 26 02:04:59 2010 said:


It depends on how much X-gal and IPTG was added to the plate, and what your definition of blue is. Pale blue (almost white) or deep blue.

For me, after spreading the cell culture, I leave it at 37 C for colonies to develop. After that I take the plate and leave it for another 24hr at 4-10 C. That is usually enough to tell the difference between white and pale blue.


I see! Thank for the info!

-hianghao-

also..keep your plates wrapped in Alu-foil. B-X-gal is light-sensitive and degrades rapidly

-fysio lab-