Team:Paris/Protocols Competent Bacteria

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Revision as of 19:18, 9 August 2009

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Use non competent bacteria (ex: MG1655) stocked in 1.5 mL LB (20% Glycerol): put a sterile tip in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml LB medium.
Over Night culture at 37°C / 200 rpm
1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL
Culture at 37°C / 200 rpm untill OD₆₀₀ reach 0.6
Fast cooling at +4°C by gently shaking the erlen in ice
Use pre-cooled centrifuge at +4°C. Centrifuge 50 mL of the culture in 50 mL falcon: +4°C / 5 min / 4000 rpm
Discard supernatant by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl₂ and mix gently the suspension by up and down
Add cold CaCl₂ QSP 20 mL and incubate 30 min / +4°C
Centrifuge the suspension : +4°C / 5 min / 4000 rpm
Discard supernatant by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl₂ and mix gently the suspension by up and down
Transform or freeze the competent cells. Freeze the competent cells in 50 µL aliquots in the 0.1M CaCl₂ medium with 15% glycerol.
After transformation, prepare a Glycerol Stock

Inoculate 50/5 ml of LB with fresh colony or dilution of an overnight culture
Grow up to OD₆₀₀ 0.5-0.8
Leave on ice 10min
Spin 40/2 ml of cell suspension, 5min at 4000rpm, 4°C/minispin 2min at 5000rpm, 4°C
Resuspend the pellet in 20/1 ml CaCl₂ 50mM (1/2 of initial Vol)
Leave on ice 10min
Spin 40/2 ml of resuspension, 5min at 4000rpm, 4°C/minispin 2min at 5000rpm, 4°C
Resuspend the pellet in 2ml/100µl CaCl₂ 50mM (1/20 of initial Vol)
Leave on ice overnight before stock at -80°C

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 ==Protocol to make competent bacteria (iGEM2007)==
-# Use non competent bacteria (ex: MG1655) stocked in 1.5 mL LB (20% Glycerol): put a sterile tip in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml LB medium.<br>Over Night culture at 37°C / 200 rpm
+====Prepare CaCl<sub>2</sub> 0.1M====
-# 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL
-# Culture at 37°C / 200 rpm untill OD<sub>600</sub> reach 0.6
-'''Prepare CaCl<sub>2</sub> 0.1M'''
 * Add 7,351g of CaCl<sub>2</sub>.2 H<sub>2</sub>O (FM 147,02) in 500 mL H<sub>2</sub>O
 * dissolve the CaCl<sub>2</sub> by mixing the suspension with the help of a magnetic stirrer
@@ Line 15: / Line 10: @@
 * Aliquotes the filtered solution: 25mL in a 50 mL Falcon. Storage at +4°C
+====Steps====
+# Use non competent bacteria (ex: MG1655) stocked in 1.5 mL LB (20% Glycerol): put a sterile tip in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml LB medium.<br>Over Night culture at 37°C / 200 rpm
+# 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL
+# Culture at 37°C / 200 rpm untill OD<sub>600</sub> reach 0.6
 # Fast cooling at +4°C by gently shaking the erlen in ice
 # Use pre-cooled centrifuge at +4°C. Centrifuge 50 mL of the culture in 50 mL falcon: +4°C / 5 min / 4000 rpm