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

From OpenWetWare

< User:RAMA RAJU B | Notebook | METABOLIC ENGINEERING | 2012 | 03(Difference between revisions)
Jump to: navigation, search
(Autocreate 2012/03/08 Entry for User:RAMA_RAJU_B/Notebook/METABOLIC_ENGINEERING)
Current revision (06:01, 10 March 2012) (view source)
(Entry title)
 
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-
==Entry title==
+
1.Revival of cultures/isolating a single colony
-
* Insert content here...
+
 +
 +
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.
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Current revision

Project name Main project page

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.


Personal tools