Difference between revisions of "Team:Paris Saclay/Experiments"

(→‎Heat shock competent cells tranformation)
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==Protocols==
 
==Protocols==
  
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===Heat shock competent cells preparation===
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===Heat shock competent cells===
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====Preparation====
 
'''Day 1''' Inoculate cells in 3.5mL LB medium.
 
'''Day 1''' Inoculate cells in 3.5mL LB medium.
 
Incubate at 37°C overnight.
 
Incubate at 37°C overnight.
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Dissolve HEPES, CaCl<sub>2</sub> and KCl in water. Adjust pH to 6.7 with KOH. Add MnCl<sub>2</sub>. Filter to sterilize and keep at 4°C.
 
Dissolve HEPES, CaCl<sub>2</sub> and KCl in water. Adjust pH to 6.7 with KOH. Add MnCl<sub>2</sub>. Filter to sterilize and keep at 4°C.
  
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===Heat shock competent cells tranformation===
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====Tranformation====
 
Add 1µL of plasmids to 50µL of competent cells (make a control tube without plasmid).  
 
Add 1µL of plasmids to 50µL of competent cells (make a control tube without plasmid).  
 
Keep tubes on ice for 30min and heat shock at 42°C for 1min.  
 
Keep tubes on ice for 30min and heat shock at 42°C for 1min.  
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Spread cells on Petri dishes in duplicate and incubate at 37°C overnight.
 
Spread cells on Petri dishes in duplicate and incubate at 37°C overnight.
  
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===Electro-competent cells preparation===
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===Electro-competent cells===
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====Preparation====
 
Inoculate 15mL of LB with 200µL of an overnight cell culture.  
 
Inoculate 15mL of LB with 200µL of an overnight cell culture.  
 
Incubate at 37°C and 180rpm until OD<sub>600nm</sub> reaches 0.6.  
 
Incubate at 37°C and 180rpm until OD<sub>600nm</sub> reaches 0.6.  
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Wash twice with 10mL of 10% glycerol.
 
Wash twice with 10mL of 10% glycerol.
 
Put cells in 200µL of 10% glycerol and use for electroporation.
 
Put cells in 200µL of 10% glycerol and use for electroporation.
 +
 +
===Transformation====

Revision as of 15:37, 6 July 2016

Experiments

Describe the experiments, research and protocols you used in your iGEM project.

What should this page contain?

  • Protocols
  • Experiments
  • Documentation of the development of your project

Protocols

Heat shock competent cells

Preparation

Day 1 Inoculate cells in 3.5mL LB medium. Incubate at 37°C overnight.

Day 2 Measure culture OD at 600nm. Dilute cells in 250mL of LB medium so OD equals to 0.12. Incubate overnight at 20°C and 180rpm.

Day 3 Measure culture OD at 600nm and dilute to obtain OD600nm=0.6. Cells must be kept at 4°C during all following steps. Put on ice for 10min and centrifuge for 10min at 3000rpm and 4°C. Discard supernatant and resuspend cells in 80mL of fresh TB buffer. Keep on ice for 10min and centrifuge for 10min at 3000rpm and 4°C. Discard supernatant again and resuspend cells in 20mL of fresh TB buffer with 7% of DMSO. Keep on ice for 10min. Aliquot cells and freeze with liquid nitrogen. Keep at -80°C.

TB buffer recipe

HEPES 10mM
MnCl2 55mM
CaCl2 15mM
KCl 250mM
KOH

Dissolve HEPES, CaCl2 and KCl in water. Adjust pH to 6.7 with KOH. Add MnCl2. Filter to sterilize and keep at 4°C.

Tranformation

Add 1µL of plasmids to 50µL of competent cells (make a control tube without plasmid). Keep tubes on ice for 30min and heat shock at 42°C for 1min. Add 500µL of LB medium into each tube and incubate at 37°C for 1h. Spread cells on Petri dishes in duplicate and incubate at 37°C overnight.

Electro-competent cells

Preparation

Inoculate 15mL of LB with 200µL of an overnight cell culture. Incubate at 37°C and 180rpm until OD600nm reaches 0.6. Centrifuge cells for 10 minutes at 4000rpm. Wash twice with 10mL of 10% glycerol. Put cells in 200µL of 10% glycerol and use for electroporation.

Transformation=