User:RAMA RAJU B/Notebook/METABOLIC ENGINEERING/2012/03/08

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1.Revival of cultures/isolating a single colony


Obtain an LB agar plate with the appropriate antibiotic. Using a sterile pipette tip, touch the bacteria growing within the punctured area of the stab culture. (A sterilized wire loop or sterile toothpick can be used in place of a sterile pipette tip.) Run this tip lightly over a section of the plate, as shown in the figure, to create streak #1. Using another sterile pipette tip, pass through streak #1 and spread the bacteria over a second section of the plate, to create streak #2. Using a third sterile pipette tip, pass through streak #2 and spread the bacteria over the last section of the plate, to create streak #3. Grow overnight in a 37 o C incubator (unless a different growth temperature is indicated on the plasmid datasheet). In the morning, single colonies should be visible. If the bacterial growth is too dense, re-streak onto a new agar plate to obtain single colonies

Recipes Luria Broth (LB) (500 mL): This recipe will make 500 mL of LB media.

Weigh ingredients below and add to 500 mL of distilled water in a bottle: 5 g Tryptone 2.5 g Yeast extract 5 g NaCl 500 mL H2O Cap bottle, but do not tighten. Autoclave using liquid cycle. Tighten lid and store at room temperature. Luria agar plates (50): This recipe will make about 50 plates (100 mm diameter).

Weigh ingredients below and add to 1L of distilled water in a 2L flask: 10 g Tryptone 5 g Yeast extract 10 g NaCl 15 g Agar 1 L H2O Cover flask tightly with foil. Autoclave using liquid cycle, then cool to 55 o C. Add appropriate volume of antibiotic. (For example, add 1 mL of a 100 mg/mL ampicillin stock to obtain a final concentration of 100 μg/mL). Pour a thin layer of LB agar into each petri dish (about 20 mL), and cover with lid immediately. Let plates cool for a few hours or overnight. Store plates in plastic bag at 4 o C.

50% glycerol (500 mL):


Add 250 mL 100% glycerol to 250 mL distilled water in a bottle. Cap bottle, but do not tighten. Autoclave using liquid cycle. Tighten lid and store at room temperature.


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