Difference between revisions of "Team:Toulouse/Experiments"
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<li><a href="#part5">- Digestion</a></li> | <li><a href="#part5">- Digestion</a></li> | ||
| − | <li><a href="#part6">- Parts have the same antibiotic resistance</a></li> | + | <ul> |
| + | <li><a href="#part6">- Parts have the same antibiotic resistance</a></li> | ||
<li><a href="#part7">- Parts have different antibiotic resistances</a></li> | <li><a href="#part7">- Parts have different antibiotic resistances</a></li> | ||
| − | <li><a href="#part8">- Ligation</a></li> | + | </ul> |
| + | <li><a href="#part8">- Ligation</a></li> | ||
<li><a href="#part9">- Transformation</a></li> | <li><a href="#part9">- Transformation</a></li> | ||
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<center><div class="subtitle" > | <center><div class="subtitle" > | ||
| − | <h3> | + | <h3>Cloning</h3><!-- CLONING --> |
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Revision as of 12:31, 1 September 2015
Experiments & Protocols
Transformation Protocol: RbCl method
Media and solution
| YETM 500mL | TFB1 200mL | TFB2 200mL |
|---|---|---|
|
|
|
Preparation of Competent Cells
- 1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37°C
- 2. Pick a single fresh colony to inoculate 5mL of YETM medium. Grow over night at 37°C.
- Do not vortex cells at any time after this point in the procedure
- 3. Dilute 1mL of culture into 50mL YETM medium prewarmed to 37°C
- Grow at 37°C for 2hr with agitation
- Volumes can be scaled up 5X and all of the 5mL overnight culture can be used
- 4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes
- 5. Centrifuge for 10 minutes at 2000rpm at 4°C. Immediately aspirate off all of the supernatant
- Do not allow cells to warm above 4°C at any time in this procedure
- 6. Resuspend cells in 10mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette
- 7. Incubate cells on ice for 5min
- 8. Repeat step 5
- 9. Resuspend cells in 2mL of ice-cold TFB2 with gentle re-pipetting. Use micropipet tip (plastic)
- 10. Incubate cells on ice for 15 minutes
- Cells may be used for transformation or frozen
- To freeze: aliquot cell in 200μL volumes into prechilled 0.5mL microfuge tube (on ice)
- Freeze immediately in liquid nitrogen
- Store cells frozen at -80°C
Transformation of Competent Cells
- 1. If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed.
Immedately place on ice for about 5 minutes - 2. Add to 1,5mL eppendorff on ice: 2-3μL iGEM plate or 1μL plasmid or 10μL ligation.
- 3. Add 100μL of competent cells and mix by gentle re-pipetting
- 4. Incubate cells on ice for 20-30 minutes
- 5. Heat shock the cells exactly 90 seconds at 42°C
- 6. Return cells on ice for 2 minutes
- 7. Add 1mL of YETM medium. Incubate at 37°C for 45-60 minutes with slow gentle shaking
- 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate overnight at 37°C
Minipreps
- 1. Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)
- 2. Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic
- 3. Let the culture grow overnight at 37°C in a shaking incubator
- 4. Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55°C
- 5. Keep the tubes at -20°C
Cloning
References
