Team:Evry/Notebook/Protocols

From 2014.igem.org

Revision as of 10:59, 17 October 2014 by NoémieBC (Talk | contribs)

IGEM Evry 2014

Notebook - Protocols

Primers



Electro-competent Pseudovibrio in Marine broth



Reagents and Materials

* Refrigerated centrifuge (4°C)
* Marine Broth 1X
*Cold glycerol 10% (4°C)

Experimental procedure

-Make a pre-culture of Pseudovibrio in 3mL of MB 1X and let it incubate overnight at 30°C with shaking.

-Relaunch the Pseudovibrio culture in 100mL of MB 1X.
-Let it grow in a 30°C shaking incubator until DO (600nm) = 1.2
-Divide the culture into two 50mL Falcon tubes.
-Wash cells with cold glycerol 10% five times
(Centrifuge at 4000g, 4°C for 10min and re-suspend):
* 50 mL (x 1)
* 25 mL (x 1)
* 5 mL (x 3)
-Re-suspend a last time washed cells in 500µL of glycerol 10%.
-Aliquot 50µL of competent cells and stock them at -80°C for maximum three weeks.

Electro-competent Pseudovibrio in M9-CASA +3%NaCl



Reagents and Materials

* Refrigerated centrifuge (4°C)
* M9-CASA+3%NaCl 1X
*Cold glycerol 10% (4°C)
*Cold sorbitol 2% (4°C)

Experimental procedure

-Make a pre-culture of Pseudovibrio in 3mL of M9-CASA+3%NaCl 1X and let it incubate overnight at 30°C with shaking.

-Relaunch the Pseudovibrio culture in 100mL of M9-CASA+3%NaCl 1X.
-Let it grow in a 30°C shaking incubator until DO (600nm) = 1.2
-Divide the culture into two 50mL Falcon tubes.
-Wash cells with cold glycerol or cold sorbitol three times
(Centrifuge at 4000g, 4°C for 10min and re-suspend):
* 50 mL (x 1)
* 25 mL (x 1)
* 5 mL (x 1)
-Re-suspend a last time washed cells in 500µL of glycerol 10%.
-Aliquot 50µL of competent cells and stock them at -80°C for maximum three weeks.

Marine broth



Composition of the medium:

text to print if image not found

After having put the wished volume of water, put 40.2g/L of the medium powder.
Dissolve sediments by warming the mixture.
Boil during one entiere minute
Autoclave the medium during 15min at 250°F.


M9 - CASA +3%NaCl



Composition of the medium:

text to print if image not found

After add correspondant quantity of the different compounds in the wished volume of water, filtrate the entiere volume obtained.
NB: This medium can't be autoclaved contain glucose and amino acids.

Preparation of antibiotic stocks

Antibiotic Stock concentration Protocol
Kanamycin 25 mg/mL Weight 0.56g of Kanamycin powder, solubilization into 20mL of miliQ water.
Vortex 5min and filtration with a 200nm filter.
Stock : -20°C (aliquots of 1mL)

Streptomycin 100 mg/mL Weight 1g of Streptomycin powder, solubilization into 20mL of miliQ water.
Vortex 5min and filtration with a 200nm filter.
Stock : -20°C (aliquots of 1mL)

Amplicilin 100 mg/mL Weight 1g of Amplicilin powder, solubilization into 20mL of miliQ water.
Vortex 5min and filtration with a 200nm filter.
Stock : -20°C (aliquots of 1mL)

Tetracyclin 15 mg/mL Weight 0.6g of Tetracyclin powder, solubilization into 20mL of ethanol 50%.
Vortex 5min and filtration with a 200nm filter.
Stock : -20°C and hiden from light(aliquots of 1mL)

Chloramphenicol 34 mg/mL Weight 0.34g of Chloramphenicol powder, solubilization into 10mL of ethanol 100%.
Vortex 5min and filtration with a 200nm filter.
Stock : -20°C and hiden from light(aliquots of 1mL)