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Team:Waterloo/Notebook/Recipes
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(New page: * Making Auto Induction Media * Making LB Media * [[Team:Waterloo/Notebook/Making SOB Media|Making SOB...) |
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| - | + | <b><u><big>Making Auto Induction (AI) Media</big></u></b><br> | |
| + | |||
| + | <b>Before you start </b><br> | ||
| + | Certain parts are on variable copy number plasmids. The plasmid is normally low copy number, but when lactose is used to induce, the plasmid becomes high copy number. Auto-Induction media causes this to happen conveniently by providing glucose and lactose. The cells will feed on glucose and replicate to high density. The glucose will then be exhausted and the lactose will induce the plasmid to become high copy number. The lactose does not induce the plasmid before it is at high density thanks to catabolite repression. <br> | ||
| + | |||
| + | <b>Materials </b><br> | ||
| + | 500 mL media bottle<br> | ||
| + | 1L beaker <br> | ||
| + | Magnetic Stirrer<br> | ||
| + | Na<sub>2</sub>HPO<sub>4</sub> <br> | ||
| + | KH<sub>2</sub>PO<sub>4</sub> <br> | ||
| + | Yeast extract <br> | ||
| + | NaCl <br> | ||
| + | Glycerol <br> | ||
| + | NaOH <br> | ||
| + | 10% glucose solution<br> | ||
| + | 8% lactose solution <br> | ||
| + | |||
| + | <b>Instructions </b><br> | ||
| + | 1. Fill 1 L beaker with 500 mL of deionized water. <br> | ||
| + | 2. Put on magnetic stirrer. <br> | ||
| + | 3. Measure and add to beaker: <br> | ||
| + | - 3g Na<sub>2</sub>HPO<sub>4</sub><br> | ||
| + | - 1.5g KH<sub>2</sub>PO<sub>4</sub> <br> | ||
| + | - 2.5g yeast extract <br> | ||
| + | - 2.5g sodium chloride <br> | ||
| + | - 3g glycerol <br> | ||
| + | 4. Adjust pH to 7.2 with NaOH using a pH-meter. <br> | ||
| + | 5. Fill the media bottle. <br> | ||
| + | 6. Autoclave with slightly open lid on slow exchange.<br> | ||
| + | 7. Filter sterilize 10% glucose solution.<br> | ||
| + | 8. Filter sterilize 8% lactose solution. <br> | ||
| + | |||
| + | At the time you are ready to prepare a culture: <br> | ||
| + | 1.Add 12.5mL of lactose solution to the autoclaved stock. <br> | ||
| + | 2.Add 2.5mL of glucose solution to the autoclaved stock. <br> | ||
| + | - If preparing 5 mL culture tubes, use 125µL, and 25µL, respectively.<br> | ||
| + | |||
* [[Team:Waterloo/Notebook/Making LB Media|Making LB Media]] | * [[Team:Waterloo/Notebook/Making LB Media|Making LB Media]] | ||
* [[Team:Waterloo/Notebook/Making SOB Media|Making SOB Media]] | * [[Team:Waterloo/Notebook/Making SOB Media|Making SOB Media]] | ||
Revision as of 21:16, 28 October 2008
Making Auto Induction (AI) Media
Before you start
Certain parts are on variable copy number plasmids. The plasmid is normally low copy number, but when lactose is used to induce, the plasmid becomes high copy number. Auto-Induction media causes this to happen conveniently by providing glucose and lactose. The cells will feed on glucose and replicate to high density. The glucose will then be exhausted and the lactose will induce the plasmid to become high copy number. The lactose does not induce the plasmid before it is at high density thanks to catabolite repression.
Materials
500 mL media bottle
1L beaker
Magnetic Stirrer
Na2HPO4
KH2PO4
Yeast extract
NaCl
Glycerol
NaOH
10% glucose solution
8% lactose solution
Instructions
1. Fill 1 L beaker with 500 mL of deionized water.
2. Put on magnetic stirrer.
3. Measure and add to beaker:
- 3g Na2HPO4
- 1.5g KH2PO4
- 2.5g yeast extract
- 2.5g sodium chloride
- 3g glycerol
4. Adjust pH to 7.2 with NaOH using a pH-meter.
5. Fill the media bottle.
6. Autoclave with slightly open lid on slow exchange.
7. Filter sterilize 10% glucose solution.
8. Filter sterilize 8% lactose solution.
At the time you are ready to prepare a culture:
1.Add 12.5mL of lactose solution to the autoclaved stock.
2.Add 2.5mL of glucose solution to the autoclaved stock.
- If preparing 5 mL culture tubes, use 125µL, and 25µL, respectively.
