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Team:Washington/Protocols/Competent
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| + | =Competent Cell Stocks= | ||
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| + | '''CCMB Transformation''' | ||
1. Plate 5ul of cells onto an antibiotic free LB/Agar plate. | 1. Plate 5ul of cells onto an antibiotic free LB/Agar plate. | ||
| - | 2. Incubate overnight at | + | 2. Incubate overnight at 37°C. |
3. Inoculate 6 colonies into 50ml SOB-Mg media in 250ml flask. | 3. Inoculate 6 colonies into 50ml SOB-Mg media in 250ml flask. | ||
| - | 4. Incubate at | + | 4. Incubate at 37°C, 200rpm, until OD at 550nm reaches 0.3, takes about four hours. |
| - | + | :Note: Allow spectrophotometer to warm up for 30 minutes before use. | |
5. Pipette the cell suspension into sterile 50ml Falcon tubes and chill on ice for 10 minutes. | 5. Pipette the cell suspension into sterile 50ml Falcon tubes and chill on ice for 10 minutes. | ||
| - | 6. Pellet the cells at 2500rpm for 15 minutes at | + | 6. Pellet the cells at 2500rpm for 15 minutes at 4°C. |
7. Decant supernatant and invert tubes to remove excess culture medium. | 7. Decant supernatant and invert tubes to remove excess culture medium. | ||
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9. Incubate on ice for 20 minutes. | 9. Incubate on ice for 20 minutes. | ||
| - | 10. Centrifuge at 2500rpm for 10 minutes at | + | 10. Centrifuge at 2500rpm for 10 minutes at 4°C. |
11. Decant supernatant and invert tubes to remove excess liquid. | 11. Decant supernatant and invert tubes to remove excess liquid. | ||
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12. Resuspend the cells in CCMB at 1/12 original volume (4ml). | 12. Resuspend the cells in CCMB at 1/12 original volume (4ml). | ||
| - | 13. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store in - | + | 13. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store in -80°C. |
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3. Add salts, allowing each to enter into solution before adding the next. | 3. Add salts, allowing each to enter into solution before adding the next. | ||
| - | 4. Add | + | 4. Add ddH<sub>2</sub>O to 1 Liter |
5. Adjust pH to 6.4 with 0.1M HCl. Do not adjust pH upward with base. | 5. Adjust pH to 6.4 with 0.1M HCl. Do not adjust pH upward with base. | ||
| - | 6. Filter and store at | + | 6. Filter and store at 4°C. |
Latest revision as of 02:52, 16 September 2011
Competent Cell Stocks
CCMB Transformation
1. Plate 5ul of cells onto an antibiotic free LB/Agar plate.
2. Incubate overnight at 37°C.
3. Inoculate 6 colonies into 50ml SOB-Mg media in 250ml flask.
4. Incubate at 37°C, 200rpm, until OD at 550nm reaches 0.3, takes about four hours.
- Note: Allow spectrophotometer to warm up for 30 minutes before use.
5. Pipette the cell suspension into sterile 50ml Falcon tubes and chill on ice for 10 minutes.
6. Pellet the cells at 2500rpm for 15 minutes at 4°C.
7. Decant supernatant and invert tubes to remove excess culture medium.
8. Resuspend the cells in 1/3 volume of CCMB (16ml) by gentle vortexing or pipetting.
9. Incubate on ice for 20 minutes.
10. Centrifuge at 2500rpm for 10 minutes at 4°C.
11. Decant supernatant and invert tubes to remove excess liquid.
12. Resuspend the cells in CCMB at 1/12 original volume (4ml).
13. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store in -80°C.
SOB-Mg Growth Medium
20g Tryptone
5g Yeast Extract
10ml 1M NaCl
2.5ml 1M KCl
Add dH2O to 1L
Autoclave
CCMB
Compound Amount/Liter Final Concentration
Potassium Acetate 10ml of 1M stock, pH7.0 10mM
Glycerol 100g 10% (w/v)
CaCl2.2H20 11.8g 80mM
MnCl2.4H20 4.0g 20mM
MgCl2.6H20 2.0g 10mM
1. Prepare 1M solution of potassium acetate, pH7.0, using K0H, filter and store frozen.
2. Prepare a solution of 10% potassium acetate, 10% glycerol.
3. Add salts, allowing each to enter into solution before adding the next.
4. Add ddH2O to 1 Liter
5. Adjust pH to 6.4 with 0.1M HCl. Do not adjust pH upward with base.
6. Filter and store at 4°C.
