Team:Paris/Protocols Competent Bacteria
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{{Template:Paris2009_menu}} | {{Template:Paris2009_menu}} | ||
- | ==Protocol to make competent bacteria== | + | ==Protocol to make competent bacteria (iGEM2007)== |
- | + | ====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 | ||
+ | # Filter the solution with a cell-culture unit of filtration | ||
+ | # 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 | ||
+ | # 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. | ||
+ | # After transformation, prepare a [[Team:Paris/Protocols_Glycerol_Stock | Glycerol Stock]] | ||
- | + | <html> | |
+ | </div> | ||
+ | <div id="paris_content_boxtop"> | ||
+ | </div> | ||
+ | <div id="paris_content"> | ||
+ | </html> | ||
- | + | ==2nd Protocol for competent bacteria== | |
- | + | # Inoculate <u>50</u>/5 ml of LB with fresh colony or <u>dilution of an overnight culture</u> | |
- | + | # Grow up to OD<sub>600</sub> 0.5-0.8 | |
- | + | # Leave on ice 10min | |
- | + | # Spin <u>40</u>/2 ml of cell suspension, <u>5min at 4000rpm, 4°C</u>/minispin 2min at 5000rpm, 4°C | |
- | + | # Resuspend the pellet in <u>20</u>/1 ml CaCl<sub>2</sub> 50mM (1/2 of initial Vol) | |
- | + | # Leave on ice 10min | |
+ | # Spin <u>40</u>/2 ml of resuspension, <u>5min at 4000rpm, 4°C</u>/minispin 2min at 5000rpm, 4°C | ||
+ | # Resuspend the pellet in <u>2ml</u>/100µl CaCl<sub>2</sub> 50mM (1/20 of initial Vol) | ||
+ | # Leave on ice overnight before stock at -80°C | ||
- | + | <html> | |
+ | </div> | ||
+ | <div id="paris_content_boxtop"> | ||
+ | </div> | ||
+ | <div id="paris_content"> | ||
+ | </html> | ||
+ | ==Protocol for competent bacteria by RbCl== | ||
+ | ====Solution for competent bacteria (by RbCl)==== | ||
+ | *Tampon I (250ml) | ||
+ | **Potassium acetate (C<sub>2</sub>H<sub>3</sub>KO<sub>2</sub> 30mM pH 5,8 0,733g | ||
+ | **RbCl 100mM 3,02g | ||
+ | **CaCl<sub>2</sub> 10mM 0,3741g | ||
+ | **MnCl<sub>2</sub>50mM 2,5g | ||
+ | **Glycerol 15% ~37,5ml | ||
+ | **QS 250ml H<sub>2</sub>O ~218,5ml | ||
+ | **Adjust with Acetic acid (CH<sub>3</sub>COOH 100%)to pH 5,8. (Remake the solution if pH beneath 5.8 | ||
+ | **Sterilization (filtration). | ||
- | |||
- | 6. | + | *Tampon II (125ml) |
+ | **MOPS 10mM pH 6.5 0,2382g | ||
+ | **RbCl 10mM 0,150g | ||
+ | **CaCl<sub>2</sub> 10mM 1,37gg | ||
+ | **Glycerol 15% ~18,75ml | ||
+ | **QS 1250ml H<sub>2</sub>O ~106.25ml | ||
+ | **Adjust pH to 6.5 with KOH 1M | ||
+ | **Sterilization (filtration) | ||
- | + | ====Steps==== | |
- | + | # Over night culture (O.N.C) | |
- | + | # Competent | |
- | + | ## 50mL of LB with 0.5mL O.N.C | |
- | + | ## Wait for 5.6 OD<sub>600</sub> | |
- | + | ## 10mn on ice | |
- | + | ## centrifuge 10mn at 4000 RPM | |
- | + | ## resuspend the bacteria pellet in 18mL Tampon I | |
- | + | ## 10mn on ice | |
+ | ## centrifuge 10mn at 4000 RPM | ||
+ | ## resuspend the bacteria pellet in 8mL Tampon II | ||
+ | ## Alicot 250µL/tube at -80°C | ||
+ | # Transformation plasmid (10pg/µl) | ||
+ | ## 250µl of RbCl competent DH5α | ||
+ | ## 12,5µl plasmid | ||
+ | ## Leave 20 min in ice | ||
+ | ## 45s at 42°C | ||
+ | ## Leave 2 min in ice | ||
+ | ## Add 1ml of hot LB (42°C) | ||
+ | ## 1h at 37 under agitation | ||
+ | ## Put on plate (LB agar + Antibiotic) | ||
+ | #culture | ||
+ | ## take 100µL and spread it on the plate (LB + Antibiotic) | ||
+ | ## centrifuge 2mn the left | ||
+ | ## take 100µL 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
- Add 7,351g of CaCl2.2 H2O (FM 147,02) in 500 mL H2O
- dissolve the CaCl2 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
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.
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
2nd Protocol for competent bacteria
- 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
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
- Over night culture (O.N.C)
- Competent
- 50mL of LB with 0.5mL O.N.C
- Wait for 5.6 OD600
- 10mn on ice
- centrifuge 10mn at 4000 RPM
- resuspend the bacteria pellet in 18mL Tampon I
- 10mn on ice
- centrifuge 10mn at 4000 RPM
- resuspend the bacteria pellet in 8mL Tampon II
- Alicot 250µL/tube at -80°C
- Transformation plasmid (10pg/µl)
- 250µl of RbCl competent DH5α
- 12,5µl plasmid
- Leave 20 min in ice
- 45s at 42°C
- Leave 2 min in ice
- Add 1ml of hot LB (42°C)
- 1h at 37 under agitation
- Put on plate (LB agar + Antibiotic)
- culture
- take 100µL and spread it on the plate (LB + Antibiotic)
- centrifuge 2mn the left
- take 100µL and spread it on the plate (LB + Antiobotic)