When X-gal goes bad - Or, How long should I incubate my plates? (Nov/27/2009 )

Hi knowledgeable ones,

I have been using a TA cloning kit from Invitrogen and TOP10 chemically competent cells to clone a 480-bp fragment of insect mtDNA into plasmid for later screening (the cloning is to separate template from an unknown number of arthropod species, amplified from bat feces using a universal arthropod primer).

I am still working hard to optimize my protocol and am still in the early stages of figuring out what will increase my ligation and transformation efficiency. Sometimes I get very few colonies per plate, sometimes the colony growth is more reasonable; but I've consistently had mostly white colonies with only a very scarce few that are a very pale blue after overnight incubation on LB/amp plates. When I did some colony screening on my latest batch of plates Weds (after overnight incubation and then putting the plates in the fridge for a couple of hours to let the color develop), I found that (1) most colonies were suspiciously white (at another lab I had way more blue colonies), and (2) most of the colonies I screened ended up having no insert!

No one in my lab has been doing blue-white screening for a little while, so I suspect the X-gal solution (of unknown concentration) that was in the freezer may have gone off. I found a sealed bottle of 100 mg X-gal -- never opened -- but it was in the fridge at +4 C, not in the freezer where it ought to have been. No one in the lab that afternoon knew anything of its age. I made it into a solution in DMSO. I tried applying some to one of the plates I had, after the fact. I ended up with a smeary mess, but it was a much bluer mess. However, with extra time in the incubator (after about 48 hours), the rest of the plates also developed a much bluer color. Not as blue as the plate I added X-gal to later, but a hell of a lot more blue than when I picked the colonies (and I could see then that many I had picked were, in fact, blue with time).

So here are my questions:

(1) Would you use an unopened vial of X-gal to make a solution, if that X-gal had been in the fridge and not the freezer? Or would you just toss it? I haven't made any new plates since making the new solution so I haven't tried it the good old-fashioned empirical way.

(2) Why would the color develop so much more after more time in the incubator? Is it OK to leave the plates in the incubator for appx 48 hrs before screening? Or is there some disadvantage to incubating for so long that I am not aware of? Given my currently low transformation efficiencies, the colonies didn't become too dense to easily pick, although I hope that this changes for me soon enough.

Thanks!
Kim

I would definitely try the 100 mg bottle of X-gal; it will probably work. I'd probably toss the unlabeled concentration bottle.

What I would really do is to switch to using S-gal, a Sigma product which produces dark black colonies rather than pale blue ones.
http://www.sigmaaldrich.com/life-science/m...s-gal-home.html

Growing your cultures for 48 hours invites satellite colony formation, due to ampicillin in the medium degrading. You should have colonies and color in 12-18 hours. Satellite colonies will not have any plasmid, since they will not be amp resistant.

As you mention, X-gal plates improve after a few hours at +4.

phage434 on Nov 27 2009, 05:44 PM said:

Hi
I'd ask another lab for the X-gal solution which is working for sure and try to repeat the experiment.
Since your problem might be very low ligation efficiency and not the X-gal solution.
Best
Michael

Michaelro on Nov 28 2009, 10:27 AM said: