I used to use ready antibiotic supplied by a company, now I need to prepare antibiotic solution for cell culture purpose from streptomycin and ampicillin powder.
What exactly do I have to do to prepare antiboitic solution and in which concentration?
You can find information on resolvent, stock solutions and working dilutions for common antibiotics in literature like "Molecular Cloning" by Maniatis or e.g. in the internet on retailer's data sheets.
According to my experience and to established protocols in our lab I would suggest you the following procedure regarding these two antibiotics:
Ampicillin:
Stock Concentration: 100 mg/mL dissolved in H2O (e.g. 1g powder in 10 mL H2O), stored at -20°C in aliquots
Working dilution: 1/1000, so you have to put 500 µL stock solution in 500 mL of your medium, for example. Your final working concentration is now 100 µg/mL.
Streptomycin:
Stock concentration:10 mg/mL dissolved in H2O (e.g. 1g powder in 100 mL H2O),
stored at -20°C in aliquots
Working dilution: 1/100, so you have to put 5 mL stock solution in 500 mL of your medium, for example. Your final working concentration here is also 100 µg/mL.
I guess the stock for streptomycin is just less concentrated, because it is less soluble in H2O than ampicillin.
Please note that not all antibiotics are dissolved in water. Chloramphenicol and tetracycline for example have to be dissolved in ethanol.
Best regards,
For bacteria cultures, I use to prepare stock of ampicillin at 100mG/mL and then dilute it into the LB-Agar at a 1/1000e dilution (100µG/mL = final concentration). I use H2Omq to reconstitue the powder. For the strepto I've never used it but I think that the principle is quite the same.
You can find information on resolvent, stock solutions and working dilutions for common antibiotics in literature like "Molecular Cloning" by Maniatis or e.g. in the internet on retailer's data sheets.
According to my experience and to established protocols in our lab I would suggest you the following procedure regarding these two antibiotics:
Ampicillin:
Stock Concentration: 100 mg/mL dissolved in H2O (e.g. 1g powder in 10 mL H2O), stored at -20°C in aliquots
Working dilution: 1/1000, so you have to put 500 µL stock solution in 500 mL of your medium, for example. Your final working concentration is now 100 µg/mL.
Streptomycin:
Stock concentration:10 mg/mL dissolved in H2O (e.g. 1g powder in 100 mL H2O),
stored at -20°C in aliquots
Working dilution: 1/100, so you have to put 5 mL stock solution in 500 mL of your medium, for example. Your final working concentration here is also 100 µg/mL.
I guess the stock for streptomycin is just less concentrated, because it is less soluble in H2O than ampicillin.
Please note that not all antibiotics are dissolved in water. Chloramphenicol and tetracycline for example have to be dissolved in ethanol.
Best regards,
Thanks Christoph and Nadia for these ideas, it is very clear to me now.
The above answers cover the topic well, the only thing I'd add is that I filter sterilize all my antibiotics that are dissolved in 50% or greater H2O but skip this step for any dissolved in absolute ethanol.
http://openwetware.org/wiki/Ampicillin
http://openwetware.org/wiki/Streptomycin
I have a new question.... is there any problem if you store antibiotic stocks at -80°C ultra freezer?? my first answer would be NO, but......
hi. could you please help me to find how to aliquot trypsin for my cell's passage?
I am preparing a medium (500mL) for bacterial culture. How much ul of Ampicillin is required for this medium please?
I would say depends what you are working on. for molecular biology and normal cell culture I use Addgenes concentrations as a guide (100ug/ml working concentration of amp, I use similar concentration for strep)
https://www.addgene.org/mol-bio-reference/
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