Describe, in detail, to a freshman undergraduate how to make 1 liter of LB + Kan (100 μg/ml final concentration) + Amp (50 μg /ml final concentration) liquid media. [Include things like how many grams of each component that you use, how much antibiotic (in ml) to add (stock solutions – 100 mg/ml ampicillin, 25 mg/ml kanamycin), and in what type of container you perform the sterilization step.] Show your calculations.

Explanation:

The objective here is to prepare  1 liter of LB + Kan (100 μg/ml final concentration) + Amp (50 μg /ml final concentration) liquid media.

Given that :

the Stock concentration of Amp: 100 mg/ml (since 1 mg/ml = 1000 μg/ml)

it is required that we convert stock concentration in  μg/ml since 100 mg/ml = 100000  μg/ml

However, using formula C₁V₁=C₂V₂ (Ampicilin),

where:

C₁ = 100000 μg/ml,

V₁=?,

C₂= 50  μg/ml,

V₂=1000 ml

100000 μg/ml × V₁ = 50  μg/ml × 1000 ml

V₁ =  50  μg/ml × 1000 ml/100000 μg/ml

V₁ =   0.5 ml

Given that:

the Stock concentration of Kan: 25 mg/ml (since 1 mg/ml = 1000 μg/ml)

it is required that we convert stock concentration in  μg/ml , 25 mg/ml = 25000 μg/ml

Now by using formula C₁V₁=C₂V₂ (Kanamycin),

C₁ = 25000 μg/ml,

V₁=?,

C₂= 100  μg/ml,

V₂=1000 ml

25000 μg/ml × V₁ = 100  μg/ml × 1000 ml

V₁ =  100  μg/ml × 1000 ml/25000 μg/ml

V₁ =   4 ml

Thus; in 1 lite of Lb+ Kan+Amp preparation;

0.5 ml of Amp & 4 ml of kanamycin is used for their stock preparation.

Finally;

Sterilization step should be carried out in flask (Clean dry glass wares) for media in an autoclave, the container size should be twice the volume of media which is prepared.