Problem in picking colonies after transformation - (Apr/30/2006 )

Hi everyone
I am facing a strange problem. whenever I do transformation, I get a lawn of colonies which is not distinguishible in the beginning as white or blue colonies after an overnight incubation at 37 degree C, but if I keep the plate for another overnight incubation at 37 C, then I get the clear beautiful white and blue colonies. But if I pick up these colonies and spread it on another plate I am not able to get the colonies. Though I tried many times, but all in vain. Is there any life span of colony in such a way that they look like colony but after a certain period of time they die. Because in the beginning whan I picked up 18 colonies only with one colony I got the growth on plate but after I did plasmid isolation and restriction analysis I found that there was no insert in the vector. So I kept on trying pick other white colonies but saw no growth next day.
However I have already successfully cloned another gene, though I was facing the same problem in picking up the colonies in that case also but luckily got the desired clone. Transformation was done with DH5 alfa . Thanks in anticipation.

-promilasharma-

Hi

How old is your antibiotic? All antibiotics degrade with time, but tetracycline and neomycin are particularly susceptable. If your antibiotic is not fresh, try making up some more and doing more transformations, fresh antibiotic should help inhibit growth of colonies without inserts.

Also you may be using too low a concentration of IPTG or X-gal , check your protocol.

-bob1-

Thanks for the suggestion. I used ampicillin which is 4 months old. I do not think this is very old but now I will use a fresh stock of ampicillin for another experiment. and see whether I get the colonies or not but the problem is, I am not able to pick the colonies for miniprep which are visible also.

-promilasharma-

well, bad antibiotics could still explain why your picked colonies do not grow and do not always give you plasmids...they would be the result of failed selection, or cells post-transformation that grew without retaining any plasmid because the antibiotic selection is weak

-aimikins-

Hi,

Im doin cloning too.Im using ampicillin. Anyway my stock is about 6 months old n im not facing any problem with my cloning. I dont think the antibiotic is the problem. Mayb u need to increase the concentration of your antibiotic , IPTG and also X-Gal.

Hope your problem dissolves away.

-leela_t-

Overgrowing colonies on Amp plates depletes the ampicillin, and will allow untransformed bacteria to grow. Your white colonies are probably untransformed bacteria which will not grow on fresh plates. These are called "satellite" colonies, because when the density of AmpR cells is low, they form a halo of smaller colonies around the resistant colony. You should not incubate your plates that long. Try these things:

* increase your Amp concentration. We use 100 ug/ml
* supplement the Amp with carbenicillin, which is more resistant to degradation
* reduce the density of your plated cells
* put the plates into the refrigerator for a few hours prior to looking for blue colonies. This helps make colonies bluer, for reasons I do not understand.
* adjust the X-gal concentration to make the blue more visible.
* switch to S-gal (Sigma) which makes the colonies dark black instead of light blue

-phage434-

Thanks for your important tips. You have mentioned to reduce the density of plated cells. Can that be one of the reason for colonies to be invisible because I centrifuged the cells before I plated them out and then suspended the cells pellet in 200 ul of LB media and then plated the whole mixture on one LB plate. Another question is, now is there any way I can use the plate or now that is useless.

-promilasharma-

another point is that if your ampicillin and has been thawed many times, it will lose activity. So a 1 month old ampicillin stock that has been frizen and thawed many times will be less active that a 6 month stock that has never been frozen.

-ML1975-

Prepare and store ampicillin in a 50% Ethanol solution, and keep at -20. You never need to thaw it.

-phage434-