How to get rid of satellite colonies? - (Jun/10/2007 )

Dear all,
I always observed a lot of satellite colonies on my plate, even when I used freshly prepared LB ampicillin plates.

I am interested to know the way to get rid of these satellite colonies. For your information, I prepared my competent cells by CaCl2 method. Is there any other better method?

Thanks a lot.

Here are a few suggestions!

1. Make sure you are using right concentration of antibiotics, increase it if you can.
2. Make sure antibiotics has not expired.
3. Change the batch of antibotics.
4. Make sure you are not adding antibiotic to very hot agar. Let it cool down to 55C.
5. Do not leave the plates in incubator for a long time after the actual colonies appear.
6. Do not spread the bacteria in a very high concentration. If not sure, just try several dilutions of your transformed cells.
7. Sometimes, using higher % agar plate may help too.

I use a bit higher conc. of antibiotics to prevent satellite colonies. Also incubate the plates for a shorter period of time.

You could also try using carbenicillin instead of ampicillin. Carbenicillin is more stable than ampicillin and so would be effective for a longer period of time. It is, however, very much more expensive.

The easiest and more effective way to avoid satellite colonies is to incubate plates no longer than 16 hours at 37C
What is your usual incubation time?

Thank you to all your replies.

I have done most of the things you all have suggested all this while. Normally, I incubate my plates for about 13 - 15 hours. Hence, I am cracking my head to figure out what had actually happened on my plates, though I have successfully got most of my constructs. I just try to get perfect plates and make my life easier when I pick colonies.

At the moment, I am suspecting the preparation of competent cells by CaCl2 method may be the answer to my observation. What do you think about this?

What is your rationale for such thinking? I can think of none. I have used all sorts of competent cells (home-made, lab-made, purchased etc), and as long as you follow everything described till now, I don't see why you should still see the satellite colonies and I don't see why changing competent cells would be helpful.

Use methicillin/ampicillin. I think it is available from Sigma. Bacteria that can grow in ampicillin can grow in met/amp, but you don't get satellite colonies.

Also, you haven't mentioned what concentration of ampicillin you are using. You need to have 100 ug/ml in your plates.

Ok, after disappeared from the forum for quite some time, finally I have tried to prepare my competent cells by using CCMB buffer method.
Strange enough, it appeared that I got no satellite colony on my plate this time.
The Ampicillin concentration that I always use is 100ug/mL.
Try to crack my head to think of a reason but I couldn't figure out.