Addgene does not regularly monitor comments posted here, so we may not see your question immediately. We’d be happy to answer any questions sent to help@addgene.org as soon as possible. Please include the name of the video along with any questions so our support team can help. Thanks!
if time is an issue - and you don't have pre-made LB agar- simply use a large clean beaker and break off the agar from plate and re-melt it in the beaker with a microwave. Cool the media in water batch in 5 min; add antibiotics, pour media into the old plates; solidify the plates in cold room for 10 min.
Thanks for watching, and thanks for the suggestion. We agree! We're planning on including title cards in future videos. We're also slowly making our way through older videos and adding chapters into TH-cam for easier navigation. You can check out the chapters on this video now!
Hi, i am doing a study on the progressive development of resistant e. coli against amoxicillin. Could you please tell me what concentration of antibiotic should I add and how much i should it increase in a margin of two weeks? Thanks!
Thanks for your question. As a plasmid repository Addgene does not perform experiments like the one you’ve described, so we cannot provide direct experimental design advice. Sorry, but good luck!
Hi, I wanted to know if we could shorten it up and combine both the antibiotic and the bacteria in 1 tube, and spread the mixture on the petri dish? Or would it be wrong to do so?
Thanks for your question! Mixing the bacteria and antibiotic directly would expose your bacteria to a much higher concentration of antibiotic than intended. Doing so might kill off most of the bacteria and impair the growth of your culture on the plate.
Hello. I'm doing some research on the effect of ampicillin and phage concentrations on E coli, but I cannot find any protocol about the selection curve. Can you please list the procedure in the reply?
Hi, me and my co-researchers are making a study on the antibacterial properties of marang peel extract against staphylococcus aureus. May I ask, if your procedure in applying your treatment on the e. coli is a standard procedure or test?
Thanks for the question! This procedure is intended for general culture of E.coli for cloning and plasmid amplification. A more common protocol can be found on our Pouring LB Agar Plates protocol: www.addgene.org/protocols/pouring-lb-agar-plates/ Since it sounds like you're using a unique test, I recommend referring to methods cited in the literature of your field.
Hi Andres. The concentration in the agar is hard to determine because it may not diffuse evenly through the agar. That's why we suggest trying a few different concentrations and seeing how it affects wild type growth. You can find more details in the linked protocol in the video description above. We've also modified this video slightly to make it less confusing. Hope this helps!
Knowing or estimating the volume of agar in the dish, I add the appropriate amount of 1000× antibiotic stock(s) to ≈400 µL sterile water or broth, spread it over the agar, and let it dry partially open for an hour. For example, for the standard 20 mL of agar in a 90-100 mm Petri dish, 20 µL of each 1000× antibiotic stock into 400 µL water, spread over the agar results in a final "1× antibiotic". I assume it has fully diffused by the 9-16 hr the plates will incubate with cells. Agar is, after all, a hydrogel. And I've never had issues with toxicity from too much antibiotic using this method. I mean, the MICs of antibiotic-resistant strains are like tenfold higher than the antibiotic concentrations used for standard selection, of course depending on copy number and expression level of the resistance gene. For example, I found little to no growth disadvantage from growing resistant E. coli in 10×, 340 mg/L chloramphenicol or 500 mg/L kanamycin.
"Hello, we haven't performed that calculation, but you might be interested in the stochastic modeling methods from Coates et al. 2018 Elife." elifesciences.org/articles/32976
Addgene does not regularly monitor comments posted here, so we may not see your question immediately. We’d be happy to answer any questions sent to help@addgene.org as soon as possible. Please include the name of the video along with any questions so our support team can help. Thanks!
if time is an issue - and you don't have pre-made LB agar- simply use a large clean beaker and break off the agar from plate and re-melt it in the beaker with a microwave.
Cool the media in water batch in 5 min; add antibiotics, pour media into the old plates; solidify the plates in cold room for 10 min.
Great video! Would be more understandable if there is a subtitle for each major procedure
Thanks for watching, and thanks for the suggestion. We agree! We're planning on including title cards in future videos. We're also slowly making our way through older videos and adding chapters into TH-cam for easier navigation. You can check out the chapters on this video now!
Hi, i am doing a study on the progressive development of resistant e. coli against amoxicillin. Could you please tell me what concentration of antibiotic should I add and how much i should it increase in a margin of two weeks? Thanks!
Thanks for your question. As a plasmid repository Addgene does not perform experiments like the one you’ve described, so we cannot provide direct experimental design advice. Sorry, but good luck!
Hi, I wanted to know if we could shorten it up and combine both the antibiotic and the bacteria in 1 tube, and spread the mixture on the petri dish? Or would it be wrong to do so?
Thanks for your question! Mixing the bacteria and antibiotic directly would expose your bacteria to a much higher concentration of antibiotic than intended. Doing so might kill off most of the bacteria and impair the growth of your culture on the plate.
Hello. I'm doing some research on the effect of ampicillin and phage concentrations on E coli, but I cannot find any protocol about the selection curve. Can you please list the procedure in the reply?
You can find some more detail in our written protocol here: www.addgene.org/protocols/over-agar-antibiotic-plating/#selectionCurve
Hi, me and my co-researchers are making a study on the antibacterial properties of marang peel extract against staphylococcus aureus. May I ask, if your procedure in applying your treatment on the e. coli is a standard procedure or test?
Thanks for the question! This procedure is intended for general culture of E.coli for cloning and plasmid amplification. A more common protocol can be found on our Pouring LB Agar Plates protocol: www.addgene.org/protocols/pouring-lb-agar-plates/
Since it sounds like you're using a unique test, I recommend referring to methods cited in the literature of your field.
What will the concentration of the antibiotic in the agar be?
Hi Andres. The concentration in the agar is hard to determine because it may not diffuse evenly through the agar. That's why we suggest trying a few different concentrations and seeing how it affects wild type growth. You can find more details in the linked protocol in the video description above. We've also modified this video slightly to make it less confusing. Hope this helps!
Knowing or estimating the volume of agar in the dish, I add the appropriate amount of 1000× antibiotic stock(s) to ≈400 µL sterile water or broth, spread it over the agar, and let it dry partially open for an hour. For example, for the standard 20 mL of agar in a 90-100 mm Petri dish, 20 µL of each 1000× antibiotic stock into 400 µL water, spread over the agar results in a final "1× antibiotic".
I assume it has fully diffused by the 9-16 hr the plates will incubate with cells. Agar is, after all, a hydrogel. And I've never had issues with toxicity from too much antibiotic using this method. I mean, the MICs of antibiotic-resistant strains are like tenfold higher than the antibiotic concentrations used for standard selection, of course depending on copy number and expression level of the resistance gene. For example, I found little to no growth disadvantage from growing resistant E. coli in 10×, 340 mg/L chloramphenicol or 500 mg/L kanamycin.
Hi! I am comparing the effect of antibiotic tetracycline on E. coli, what would be the estimate ratio of antibiotic to Bacteria in the petri dish
"Hello, we haven't performed that calculation, but you might be interested in the stochastic modeling methods from Coates et al. 2018 Elife." elifesciences.org/articles/32976
I think that you must add 8 ul from carb not 80 ul