User:RAMA RAJU B/Notebook/METABOLIC ENGINEERING/2012/03/08: Difference between revisions
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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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Revision as of 03:01, 10 March 2012
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1.Revival of cultures/isolating a single colony
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):
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