Question

Describe, in detail, 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.

Answer 1

For 1 liter of LB media Preparation- Lb stands for Luria Bertani

List of components

Tryptone-10gm

Yeast extract/beef extract- 5gm

Nacl- 10gm

The final volume of distilled water- 1000ml/1 liter

• Add all the components in the 800 ml of distilled water in reagent bottle or conical flask and finally make up the volume to 1 liter.
• Prepared media then mixed with slight heating.
• pH should be adjusted to 7.
• The bottle or the flask then sealed with a cap or cotton plug and then cover them for autoclaving.
• Autoclave the media at 121ºC for 15min at 15psi pressure.
• After autoclaving, cool down the media and then antibiotics will be added to the media.
• Now the LB media is prepared for use.

Kanamycin calculation

Following formula will be used

C1xV1= C2xV2,

C1= concentration of stock solution

V1= volume of stock solution

C2= concentration of the working solution

V2= volume of the working solution

Firstly, all the calculations will be performed using the same units. Therefore convert in to same unit

such as for 100μg is equal to 0.1mg

100 mg still the same

1 liter is equal to 1000 ml

Now put it in the formula

C1xV1= C2xV2

100x V1= 0.1x1000ml

V1= 1 ml

Ampicillin calculation

Also with the same formula and method

C1xV1= C2xV2

25x V1= 0.05x1000,

V1= 2ml

• For the sterilization of liquid media without antibiotics autoclave will be used, which is moist heat sterilization.
• But, when antibiotic is used in the media, membrane filter is used to sterilize the media.
• Antibiotics solution is prepared and then filtered with 0.45 and 0.2μ membrane filters.
• Now this antibiotics solution is ready to use in media.