Adding Anitbiotic to Cooled Agar Plates - (May/12/2008 )

So I'm working on creating clones for 75 samples and have finished samples 1 - 20.

However, I think there is something wrong with the antibiotic in my plates (Im using Ampicillin and the TOPO TA kit for cloning) - I'm getting clones on my negative control plates, even when I plate cells that have been transformed with just water and no vector whatsoever.

I'm not sure if this is a result of the plates being too old (early april), bad antibiotic, or inactivation by heat when making the plates.

I'm doing a test today of my remaining plates, but I was wondering if there is any way I can salvage these plates if I DO find that there is something wrong with their antibiotic content. Can I add antibiotic to the surface of a cooled agar plate?


Any advice is appreciated!

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I'll say your plates might be a bit too old, and the ampicillin has worn off. If you don’t want to make new plates, you can definitely add extra antibiotics to these ones. Prepare your antibiotic at the appropriate concentration and spread it on the plate. Let it soak a couple of hours and then plate your bacteria as normal. It works! If you are using blue/white colony screening, i.e. X-gal and IPTG, you can add your antibiotic at the same time as these 2, just remember to let them be absorbed by the agar before you plate the bugs.

Good Luck!

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Early April plates, stored at 4C, should still work fine. What concentration of Amp are you using? We use 100 ug/ml. To add antibiotic to the plate, I'd recommend adding the amount of antibiotic for 25 ml (volume of the plate) to 250 ul of water, then spreading it on the plate, letting it soak in. For our ampicillin concentration, this would be a solution of 10 mg/ml. You could test your plates by putting a non-resistant strain on them (no transformation at all).

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Yep, they were stored at 4C - so I'm not sure what went wrong. I however, did not prepare the Amp solution from a stock (a prep person did) - so I'm not sure about it's age/origin. I did plate some competent cells on a plate from each of my batches today - so I'll see what happens with that.

I've been using 50 ng/ml Amp - I add 500 ul of this to 500 mls of Agar, then pour ~20 plates from this.

So this works out to 25 ul of antibiotic per plate - so maybe I'll just add 225 ul of water to that and spread? How does that sound?

Thanks for your help!

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Thanks so much! What would you think an appropriate drying time is? Ive noticed any extra liquid on the surface of the plate makes spreading my transformations a bit difficult. Would overnight be best?

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If the vector is a high copy plasmid, you could use ampicillin up to 100 ug/ml. That could reduce the background. I suggest that if you would spread the antibiotic before lunch and leave it on the bench, the plates should have dried in the afternoon.

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Your working stock is 50ng/ml ?

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Your ampicillin stock concentration is virtually non-existent. The ampicillin stock should be 50 mg/mL not 50 ng/mL - that's 10^6 less concentrated than it needs to be. That would explain your water only colonies, i'm surprised you didn't get more contamination growing there. Make a 50 mg/mL stock and then add 500 uL of this to 500 mL of agar (1:1000) and you'll get a final concentration of 50 ug/mL - which is what you need.

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You can dry the plates inverted in an incubator for a couple of hours. When you plate out your cells, there's still plenty of liquid remaining.

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