Team:Brasil-SP/Notebook
From 2014.igem.org
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+ | <h1> <strong>Lab Protocols</strong> </h1> | ||
+ | |||
+ | <h2> Competent Cell with Calcium Chloride </h2> | ||
+ | <ol> | ||
+ | <li><h3>Materials</h3></li> | ||
+ | <ul> | ||
+ | <li> Solid LB medium - 1 plate </li> | ||
+ | <li> Liquid LB medium </li> | ||
+ | <li> 2 250ml centrifuge tubes </li> | ||
+ | <li> Centrifuge </li> | ||
+ | <li> Cell sample (DH5-alpha in our case) </li> | ||
+ | <li> Autoclave </li> | ||
+ | <li> Inoculation loop </li> | ||
+ | <li> Liquid nitrogen </li> | ||
+ | <li> 37°C oven </li> | ||
+ | <li> CaCl<sub>2</sub>:</li> | ||
+ | <ul> | ||
+ | <li> 60mM CaCl<sub>2</sub> </li> | ||
+ | <li> 10mM HEPES </li> | ||
+ | <li> 15% glicerol </li> | ||
+ | <li> H<sub>2</sub>O to 100ml </li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | <li><h3> Method </h3> </li> | ||
+ | <p><strong>Day 1</strong></p> | ||
+ | <ol> | ||
+ | <li> Risk the LB plate with the cell sample (DH5-alpha) </li> | ||
+ | <li> Incubate the plate at 37°C overnight </li> | ||
+ | </ol> | ||
+ | <p><strong>Day 2</strong></p> | ||
+ | <ol> | ||
+ | <li> Sterilize in the Autoclave </li> | ||
+ | <ul> | ||
+ | <li> 100ml of liquid LB medium </li> | ||
+ | <li> 100 ml of CaCl<sub>2</sub> | ||
+ | <li> 2 250ml centrifuge tubes </li> | ||
+ | </ul> | ||
+ | <li> Prepare a 6ml LB inoculum with a DH5-alpha colony and incubate at 37°C/250rpm overnight</li> | ||
+ | </ol> | ||
+ | <p><strong>Day 3</strong></p> | ||
+ | <ol> | ||
+ | <li>Add 2ml of the inoculum in 100ml of liquid LB medium. Incubate at 37°C/250rpm until the OD reaches 0,375</li> | ||
+ | <li> Distribute the volume in 2 250ml centrifuge tubes (pre chilled) and spin at 10000rpm/7min/4°C</li> | ||
+ | <li> Discard the supernatant and ressuspend the pellet in 10ml of CaCl<sub>2</sub> solution</li> | ||
+ | <li> Spin the tubes at 7000rpm/7min/4°C</li> | ||
+ | <li> Discard the supernatant and ressuspend the pellet in 10ml of CaCl<sub>2</sub> solution. Incubate 30min on ice</li> | ||
+ | <li> Spin at 7000rpm/7min/4°C</li> | ||
+ | <li> Discard the supernatant and ressuspend the pellet in 2ml of the CaCl<sub>2</sub> solution</li> | ||
+ | <li> Distribute the 2ml in 500ul tubes adding 50ul in each.</li> | ||
+ | <li> Freeze the samples in liquid nitrogen </li> | ||
+ | <li> Stock at -80°C </li> | ||
+ | </ol> | ||
+ | </ol> | ||
+ | |||
+ | |||
+ | |||
</html> | </html> |
Revision as of 19:47, 22 August 2014
WELCOME TO iGEM 2014!Your team has been approved and you are ready to start the iGEM season!
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Notebook | ||||||||||||
You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well. |
Lab Protocols
Competent Cell with Calcium Chloride
Materials
- Solid LB medium - 1 plate
- Liquid LB medium
- 2 250ml centrifuge tubes
- Centrifuge
- Cell sample (DH5-alpha in our case)
- Autoclave
- Inoculation loop
- Liquid nitrogen
- 37°C oven
- CaCl2:
- 60mM CaCl2
- 10mM HEPES
- 15% glicerol
- H2O to 100ml
Method
- Risk the LB plate with the cell sample (DH5-alpha)
- Incubate the plate at 37°C overnight
- Sterilize in the Autoclave
- 100ml of liquid LB medium
- 100 ml of CaCl2
- 2 250ml centrifuge tubes
- Prepare a 6ml LB inoculum with a DH5-alpha colony and incubate at 37°C/250rpm overnight
- Add 2ml of the inoculum in 100ml of liquid LB medium. Incubate at 37°C/250rpm until the OD reaches 0,375
- Distribute the volume in 2 250ml centrifuge tubes (pre chilled) and spin at 10000rpm/7min/4°C
- Discard the supernatant and ressuspend the pellet in 10ml of CaCl2 solution
- Spin the tubes at 7000rpm/7min/4°C
- Discard the supernatant and ressuspend the pellet in 10ml of CaCl2 solution. Incubate 30min on ice
- Spin at 7000rpm/7min/4°C
- Discard the supernatant and ressuspend the pellet in 2ml of the CaCl2 solution
- Distribute the 2ml in 500ul tubes adding 50ul in each.
- Freeze the samples in liquid nitrogen
- Stock at -80°C
Day 1
Day 2
Day 3