Team:Paris/Protocols Competent Bacteria

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==Protocol to make competent bacteria==
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==Protocol to make competent bacteria (iGEM2007)==
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1. 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
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====Prepare CaCl<sub>2</sub> 0.1M====
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# 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
 +
# Filter the solution with a cell-culture unit of filtration
 +
# Aliquotes the filtered solution: 25mL in a 50 mL Falcon. Storage at +4°C
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2. 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL
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====Steps====
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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.<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
 +
# Discard supernatant by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl<sub>2</sub> and mix gently the suspension by up and down
 +
# Add cold CaCl<sub>2</sub> 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 CaCl<sub>2</sub> and mix gently the suspension by up and down
 +
# [[Team:Paris/Protocols_Transform | Transform]] or freeze the competent cells. Freeze the competent cells in 50 µL aliquots in the 0.1M CaCl<sub>2</sub> medium with 15% glycerol.
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# After transformation, prepare a [[Team:Paris/Protocols_Glycerol_Stock | Glycerol Stock]]
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3. Culture at 37°C / 200 rpm untill OD<sub>600</sub> reach 0.6
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==2nd Protocol for competent bacteria==
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Prepare CaCl<sub>2</sub> 0.1M.
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# Inoculate <u>50</u>/5 ml of LB with fresh colony or <u>dilution of an overnight culture</u>
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* 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
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# Grow up to OD<sub>600</sub> 0.5-0.8
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* dissolve the CaCl<sub>2</sub> by mixing the suspension with the help of a magnetic stirrer
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# Leave on ice 10min
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* Filter the solution with a cell-culture unit of filtration
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# Spin <u>40</u>/2 ml of cell suspension, <u>5min at 4000rpm, 4°C</u>/minispin 2min at 5000rpm, 4°C
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* Aliquotes the filtered solution: 25mL in a 50 mL Falcon. Storage at +4°C
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# Resuspend the pellet in <u>20</u>/1 ml CaCl<sub>2</sub> 50mM (1/2 of initial Vol)
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# Leave on ice 10min
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# Spin <u>40</u>/2 ml of resuspension, <u>5min at 4000rpm, 4°C</u>/minispin 2min at 5000rpm, 4°C
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# Resuspend the pellet in <u>2ml</u>/100µl CaCl<sub>2</sub> 50mM (1/20 of initial Vol)
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# Leave on ice overnight before stock at -80°C
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4. Fast cooling at +4°C by gently shaking the erlen in ice
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5. Use pre-cooled centrifuge at +4°C. Centrifuge 50 mL of the culture in 50 mL falcon: +4°C / 5 min / 4000 rpm
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==Protocol for competent bacteria by RbCl==
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6. Discard supernatant by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl<sub>2</sub> and mix gently the suspension by up and down
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====Solution for competent bacteria (by RbCl)====
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*Tampon I (250ml)
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**Potassium acetate (C<sub>2</sub>H<sub>3</sub>KO<sub>2</sub> 30mM pH 5,8 0,733g
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**RbCl 100mM 3,02g
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**CaCl<sub>2</sub> 10mM 0,3741g
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**MnCl<sub>2</sub>50mM 2,5g
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**Glycerol 15% ~37,5ml
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**QS 250ml H<sub>2</sub>O ~218,5ml
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**Adjust with Acetic acid (CH<sub>3</sub>COOH 100%)to pH 5,8. (Remake the solution if pH beneath 5.8
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**Sterilization (filtration).
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7. Add cold CaCl<sub>2</sub> QSP 20 mL and incubate 30 min / +4°C
 
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8. Centrifuge the suspension : +4°C / 5 min / 4000 rpm
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*Tampon II (125ml)
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**MOPS 10mM pH 6.5 0,2382g
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**RbCl 10mM 0,150g
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**CaCl<sub>2</sub> 10mM 1,37gg
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**Glycerol 15% ~18,75ml
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**QS 1250ml H<sub>2</sub>O ~106.25ml
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**Adjust pH to 6.5 with KOH 1M
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**Sterilization (filtration)
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9. Discard supernatant by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl<sub>2</sub> and mix gently the suspension by up and down
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====Steps====
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# Over night culture (O.N.C)
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10. [[Team:Paris/Protocols_Transform | Transform]] or freeze the competent cells. Freeze the competent cells in 50 µL aliquots in the 0.1M CaCl<sub>2</sub> medium with 15% glycerol.
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# Competent
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## 50mL of LB with 0.5mL O.N.C
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11. After transformation, prepare a [[Team:Paris/Protocols_Glycerol_Stock | Glycerol Stock]]
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## Wait for 5.6 OD<sub>600</sub>
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## 10mn on ice
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## centrifuge 10mn at 4000 RPM
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## resuspend the bacteria pellet in 18mL Tampon I
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## 10mn on ice 
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## centrifuge 10mn at 4000 RPM
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## resuspend the bacteria pellet in 8mL Tampon II
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## Alicot 250&micro;L/tube at -80°C
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# Transformation plasmid (10pg/µl)
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## 250µl of RbCl competent DH5&alpha;
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## 12,5µl plasmid
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## Leave 20 min in ice
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## 45s at 42°C
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## Leave 2 min in ice
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## Add 1ml of hot LB (42°C)
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## 1h at 37 under agitation
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## Put on plate (LB agar + Antibiotic)
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#culture
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## take 100&micro;L and spread it on the plate (LB + Antibiotic)
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## centrifuge 2mn the left
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## take 100&micro;L and spread it on the plate (LB + Antiobotic)

Latest revision as of 20:01, 9 August 2009

Contents

Protocol to make competent bacteria (iGEM2007)

Prepare CaCl2 0.1M

  1. Add 7,351g of CaCl2.2 H2O (FM 147,02) in 500 mL H2O
  2. dissolve the CaCl2 by mixing the suspension with the help of a magnetic stirrer
  3. Filter the solution with a cell-culture unit of filtration
  4. Aliquotes the filtered solution: 25mL in a 50 mL Falcon. Storage at +4°C

Steps

  1. 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
  2. 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL
  3. Culture at 37°C / 200 rpm untill OD600 reach 0.6
  4. Fast cooling at +4°C by gently shaking the erlen in ice
  5. Use pre-cooled centrifuge at +4°C. Centrifuge 50 mL of the culture in 50 mL falcon: +4°C / 5 min / 4000 rpm
  6. 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
  7. Add cold CaCl2 QSP 20 mL and incubate 30 min / +4°C
  8. Centrifuge the suspension : +4°C / 5 min / 4000 rpm
  9. 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
  10. Transform or freeze the competent cells. Freeze the competent cells in 50 µL aliquots in the 0.1M CaCl2 medium with 15% glycerol.
  11. After transformation, prepare a Glycerol Stock

2nd Protocol for competent bacteria

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

Protocol for competent bacteria by RbCl

Solution for competent bacteria (by RbCl)

  • Tampon I (250ml)
    • Potassium acetate (C2H3KO2 30mM pH 5,8 0,733g
    • RbCl 100mM 3,02g
    • CaCl2 10mM 0,3741g
    • MnCl250mM 2,5g
    • Glycerol 15% ~37,5ml
    • QS 250ml H2O ~218,5ml
    • Adjust with Acetic acid (CH3COOH 100%)to pH 5,8. (Remake the solution if pH beneath 5.8
    • Sterilization (filtration).


  • Tampon II (125ml)
    • MOPS 10mM pH 6.5 0,2382g
    • RbCl 10mM 0,150g
    • CaCl2 10mM 1,37gg
    • Glycerol 15% ~18,75ml
    • QS 1250ml H2O ~106.25ml
    • Adjust pH to 6.5 with KOH 1M
    • Sterilization (filtration)

Steps

  1. Over night culture (O.N.C)
  2. Competent
    1. 50mL of LB with 0.5mL O.N.C
    2. Wait for 5.6 OD600
    3. 10mn on ice
    4. centrifuge 10mn at 4000 RPM
    5. resuspend the bacteria pellet in 18mL Tampon I
    6. 10mn on ice
    7. centrifuge 10mn at 4000 RPM
    8. resuspend the bacteria pellet in 8mL Tampon II
    9. Alicot 250µL/tube at -80°C
  3. Transformation plasmid (10pg/µl)
    1. 250µl of RbCl competent DH5α
    2. 12,5µl plasmid
    3. Leave 20 min in ice
    4. 45s at 42°C
    5. Leave 2 min in ice
    6. Add 1ml of hot LB (42°C)
    7. 1h at 37 under agitation
    8. Put on plate (LB agar + Antibiotic)
  4. culture
    1. take 100µL and spread it on the plate (LB + Antibiotic)
    2. centrifuge 2mn the left
    3. take 100µL and spread it on the plate (LB + Antiobotic)

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