6 replies to this topic

[hianghao]

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?

[perneseblue]

hianghao, on 24 September 2010 - 04:12 PM, 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.

[hianghao]

perneseblue, on 24 September 2010 - 05:06 PM, said:

hianghao, on 24 September 2010 - 04:12 PM, 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.

[perneseblue]

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.

[phage434]

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.

[hianghao]

perneseblue, on 25 September 2010 - 06:04 PM, 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!

[fysio lab]

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