Reason for white colonies on outer rim of plates - (Aug/25/2006 )
Hi....
I recently did a cloning of a gene into a vector and attempted to do a blue/white screening. The plates were LB/Ampicillin plates were provided and spreading with IPTG with X-gal before the bacterial suspension (contaning the ligated product) was plated.
After incubation, it was observed that there were very few white colonies on the plate, and if there were the colonies were all gathered on the outer rim of the plates.
Could it be due to an insufficient amount of ampicillin on the rim of the plates or is there any other reason?
Thanks...
I recently did a cloning of a gene into a vector and attempted to do a blue/white screening. The plates were LB/Ampicillin plates were provided and spreading with IPTG with X-gal before the bacterial suspension (contaning the ligated product) was plated.
After incubation, it was observed that there were very few white colonies on the plate, and if there were the colonies were all gathered on the outer rim of the plates.
Could it be due to an insufficient amount of ampicillin on the rim of the plates or is there any other reason?
Thanks...
Hi,
Try adding the ampicillin (or whatever antibiotic) with the IPTG and X-Gal to the medium when it has cooled down to 65-60 degrees celcius, swirl the bottle a few times to mix it well, and then pour the plates. I've seen many people streaking antibiotics / IPTG / X-Gal on plates but there's no way of telling if they are streaked out well. If you add them to your medium at 65-60 degrees and then pour the plates they will degrade a little bit, but the distribution throughout the plates should be uniformly. This has always worked for me
cheers, Ite
yes I agree with Ite Teune , it might be mistake of mixing the antibiotic in media or else spreading
the antibiotic into a plate only that may be the possible reasons
Hi,
When you spread the IPTG and X-gal, you could have missed the places esp near the rim of the plate. From what you described, I think you might have self ligation problem.
What type of cloning you're working on? T/A cloning or sticky ends (single or double digest)? Anyway, if it's T/A, it shouldn't be a problem with self-ligation (only minimal).
If you're working on sticky ends ligation, do ensure that you follow the following...
(1). Gel isolate your digested vector. That'll ensure that undigested vector is not included in your ligation rxn later. If there's minute quantity of undigested vector in your ligation rxn, transformation will yield lots of blue colonies and you'll have a task to choose the white from the blues.
(2) If you're working with single cut, better do dephosphorylation on the vector. Alternatively, you could just add lots of insert per vector ratio but I'd prefer the former.
Bests,
i frequently find more whites on the periphery rather than the middle, and sometimes when the transformation efficiency is very high, i find it easier to pick up single whites from the periphery.
as a case in point, i did a miniprep a couple of days ago on bacteria from colonies up from the plate periphery, checked for the insert, and found that 5 out of the 5 that i checked, carried the insert...
- viv
If these colonies are on the rim of the plate, could it be that your spreader is not getting the x-gal spread all the way to the edge, so there is no blue/white selection in this area?
befuddled is apparently not getting any white colonies in the centre, hence the likelihood of the iptg/x-gal not spreading.
in my case, blue/white selection is certainly there. blue colonies are also at the periphery, in numbers greater than the white ones, but the white colonies are more here than in comparison to the centre of the plate.
- viv
* but where is befuddled...there are no posts from his/her end after the first!!
I recently did blue/white screeing on LB Amp plated with diffused with xgal. Got a nice mix of blue and white colonies. Picked colonies I wanted and stored the plated at 4oC (parafilmed shut in a laminar flow hood). 2 weeks later I found round pink colonies on the plates!!! Has anyone ever seen this before or am I just dirty?!
More to the the point if there is a large number of small satelite colonies, it may have to do with the concentration of ampicillan. I would use 150ul of 100mg/ml amp per 100ml of LB agar. I think you could probably go higher than that to ensure totally successful transformants
Yes, I have seen pink, red, yellow, orange, beige and bright red colonies before on my LB+ Amp plates. (Never seen purple before though)
Unfortunately, it isn't a new bit of behaviour of the LacZ/X-gal/IPTG system. You're just plain dirty. 
As for the satelite colonies, the Amp concentration 150ug/ml is very high. I think it would be better off to either use a shorter incubation time, ie catch the plates before the satelites appear. Or use carbenecilin, which is more resistent to degredation. I don't like the idea of throwing around high concentration of Amp into the environment.
If you are going to add the ampicillin to the LB prior to pouring, add it directly before pouring the plates. If the media is too hot, it denatures the ampicillin and you will get massive amounts of satellites.