Bacterial growth in agar plate with ampicillin - (Jan/25/2010 )
Hello everyone
I've again a problem in cloning during transformation.
My agar plates contain a concentration of 100ug/ml of ampicillin. After ligation, I did transformation and then overnight incubation at 37 degree C. There were only 10/15 colonies on plates with ligated sample after O/N culture. I took the plates out from 37 and stored them at 4 degree C. After 2 days of storage at 4 degree C, I have many small colonies on plates. The colonies are every where on agar (thus there is no satellite colony). Is this problem with ampicillin? Or could be the problem with undigested vector?
Waiting for any suggestions.
Thanks!
The Question on Jan 25 2010, 02:36 PM said:
I've again a problem in cloning during transformation.
My agar plates contain a concentration of 100ug/ml of ampicillin. After ligation, I did transformation and then overnight incubation at 37 degree C. There were only 10/15 colonies on plates with ligated sample after O/N culture. I took the plates out from 37 and stored them at 4 degree C. After 2 days of storage at 4 degree C, I have many small colonies on plates. The colonies are every where on agar (thus there is no satellite colony). Is this problem with ampicillin? Or could be the problem with undigested vector?
Waiting for any suggestions.
Thanks!
Hi,
Well, I think there is some contamination, possibly of some culture which is ampicillin resisitant (considering that your ampicillin is fine)
I do not know how many times have you done this expt and got the same result.
If you have done it only once, then you should repeat the expt. with controls like, along with spreaded plate, incubate a plain plate (with amp) and see if there is any growth in it after 2 days. If yes, your amp has degraded. It is time to make fresh plates. Amp plates shud not be stored for more than one month.
Regarding, vector you must ensure on gel before cloning that vector and insert are digested properly. For vector you must have done SAP treatment. So there is little chance that uncloned vector/undigested vector would contribute to the colonies and even there is, it should come in the first 24 hours only.
Also, a thumb rule is to do, a null vector transformation as well along with vector+insert, in separate comp cells. You should get more colonies in vector+insert.
Hope this helps.
Best regards,
Kaushik
KAUSHIK THAKKAR on Jan 25 2010, 11:59 AM said:
The Question on Jan 25 2010, 02:36 PM said:
I've again a problem in cloning during transformation.
My agar plates contain a concentration of 100ug/ml of ampicillin. After ligation, I did transformation and then overnight incubation at 37 degree C. There were only 10/15 colonies on plates with ligated sample after O/N culture. I took the plates out from 37 and stored them at 4 degree C. After 2 days of storage at 4 degree C, I have many small colonies on plates. The colonies are every where on agar (thus there is no satellite colony). Is this problem with ampicillin? Or could be the problem with undigested vector?
Waiting for any suggestions.
Thanks!
Hi,
Well, I think there is some contamination, possibly of some culture which is ampicillin resisitant (considering that your ampicillin is fine)
I do not know how many times have you done this expt and got the same result.
If you have done it only once, then you should repeat the expt. with controls like, along with spreaded plate, incubate a plain plate (with amp) and see if there is any growth in it after 2 days. If yes, your amp has degraded. It is time to make fresh plates. Amp plates shud not be stored for more than one month.
Regarding, vector you must ensure on gel before cloning that vector and insert are digested properly. For vector you must have done SAP treatment. So there is little chance that uncloned vector/undigested vector would contribute to the colonies and even there is, it should come in the first 24 hours only.
Also, a thumb rule is to do, a null vector transformation as well along with vector+insert, in separate comp cells. You should get more colonies in vector+insert.
Hope this helps.
Best regards,
Kaushik
Hi Kaushik
Thank you for the response.
It seems the Ampicillin I've been using is degraded. The only the thing I didn't understand is why those small colonies don't appear immediately after O/N incubation but after longer duration than O/N.
Hi,
Perhaps, at the moment there is no anwer to that question. Thats the way Science is..!!
Unless you repeat and get the same results.
Best Luck.
Cheers
Kaushik
I think you might be dismissing satellite colonies too quickly. Streak a well-isolated small colony to a fresh amp plate and see if it's truely amp resistant.
The best solution would be to get your transformants off the transformation plate by streaking them to a fresh plate as soon as you can, rather than storing the transformation plate.
