I want to pour out LB agar plates for transforming some constructs that have both ampicillin and chloramphenicol resistance markers. The concentration of ampicillin is 50mg/ml and that of chloramphenicol is 30mg/ml. I have 500ml of LB agar. I always add 500ul of ampicillin when making amp plates. However, in this case, what volume of chloramphenicol would be best advised? I do know that the volume is much lesser than for ampicillin. Thank you.
1mL of each antibiotic stock for every 1 liter of culture for each antibiotic should be fine.
So for 500mL of agar, add 0.5mL (500µl) of each antibiotic stock solution. We usually use Amp at twice the concentration you do though (100µg/mL final concentration).
Make sure you add it when the solution has cooled to 65˚C and that the antibiotic stocks are room temp before adding, then mix well to evenly distribute it before pouring plates.
Just something to remember, both these antibiotics are bacteriostatic, so they'll keep bacteria from growing, but they won't actually kill off the bacteria without your plasmid(s). If you leave your plates long enough (or have a very extended growth cycle) the antibiotics will degrade and things will start to grow that don't have your plasmids, so dispose of them quickly after use.
Normally we use 30 ug/ml of Chloram
you can use the equation; C1V1=C1V2 to calculate the volume you have to add.
For example, your stock concentration of chloram = 30mg/ml or 30000 ug/ml (I supposed it to be C1). The last concentration you need is 30 ug/ml (C2) and broth volume 500 ml (V2). So you can calculate V1 which is ;
30000 ug/ml X V2 = 30 ug/ml X 500 ml
Then V1= 0.5 ml of Chlorramphenical for 500 ml broth to get 30 ug/ml
1mL of each antibiotic stock for every 1 liter of culture for each antibiotic should be fine.
So for 500mL of agar, add 0.5mL (500µl) of each antibiotic stock solution. We usually use Amp at twice the concentration you do though (100µg/mL final concentration).
Make sure you add it when the solution has cooled to 65˚C and that the antibiotic stocks are room temp before adding, then mix well to evenly distribute it before pouring plates.
Just something to remember, both these antibiotics are bacteriostatic, so they'll keep bacteria from growing, but they won't actually kill off the bacteria without your plasmid(s). If you leave your plates long enough (or have a very extended growth cycle) the antibiotics will degrade and things will start to grow that don't have your plasmids, so dispose of them quickly after use.
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