Team:UNICAMP-EMSE Brazil/protocols/chimiocompetent prepare
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==<font size="5" color=#086A87>Preparation of Chemically competent ''E. coli'' cells== | ==<font size="5" color=#086A87>Preparation of Chemically competent ''E. coli'' cells== | ||
</font> | </font> | ||
- | |||
+ | : | ||
+ | == A) Lab materials and solutions == | ||
+ | |||
+ | :'''1) TYM (for 1 L)''' | ||
+ | :Tryptone - 20 g | ||
+ | :Yeast extract - 5 g | ||
+ | :NaCl - 5.8 g | ||
+ | :MgSO4.7H2O - 2.5 g | ||
+ | ::*AUTOCLAVE | ||
+ | ::Prepare more than 1 Erlenmeyer for each volume (for security reasons) | ||
+ | |||
+ | |||
+ | :'''2) TFB1 and TFB2''' (storage under refrigeration) | ||
+ | '''TFB1 for 1L for 200 mL''' | ||
+ | KOAC (Potassium acetate) - 0.03M - 2.94 g → 0.59 g | ||
+ | MnCl2.4H2O - 0.05M - 9.90 g → 1.98 g | ||
+ | KCl - 0.10M - 7.46 g → 1.49 g | ||
+ | CaCl2.2H2O - 0.01M - 1.47 g → 0.29 g | ||
+ | Glycerol (glycerin) - 15%(m/V) - 150 mL → 30 mL | ||
+ | :*100 ml of this solution will be used in the procedure | ||
+ | :*Filtration required (0.45µm filters) – In the flux! (for sterilization) | ||
+ | |||
+ | |||
+ | |||
+ | '''TFB2 for 1L for 200 mL''' | ||
+ | MOPS - 0.01M - 2.04 g → 0.41 g | ||
+ | KCl - 0.01M - 0.75 g → 0.15 g | ||
+ | CaCl2.2H2O - 0.075M - 11.03 g → 2.21 g | ||
+ | Glycerol (glycerin) - 15%(m/V) - 150 mL → 30 mL | ||
+ | :*Adjust the pH to 7 with KOH | ||
+ | :*Filtration required (0.45µm filters) – In the flux! (for sterilization) | ||
+ | |||
+ | |||
+ | :'''3) 1 culture plate with solid LB medium WITHOUT ANTIBIOTIC !''' | ||
+ | : To grow the colonies from the stock of competent cells | ||
+ | |||
+ | |||
+ | |||
+ | : | ||
+ | == B) Material to be checked or prepared in advance == | ||
+ | |||
+ | :'''1-''' Prepare the solutions / Place Eppendorf and Falcon in the freezer | ||
+ | |||
+ | :'''2-''' Make reservation for the following equipments: | ||
+ | :- Incubator 37°C (for colonies growth in the culture plate) | ||
+ | :- Shaker 37°C (for cells growth) | ||
+ | :- Spectrophotometer (measures at OD600) | ||
+ | :- Table centrifuge (for 50 mL Falcon) | ||
+ | :- Laminar Flux hood | ||
+ | : | ||
+ | == C) Proceedings == | ||
+ | |||
+ | :'''1) 1st day:''' Inoculate cells in the with solid LB medium without antibiotic (37°C) –overnight (inoculate by the end of the afternoon) | ||
+ | |||
+ | |||
+ | :'''2) 2nd day:''' Select colonies and let them grow overnight in a 40 mL Erlenmyer with TYM (shaker 37°C, 200 rpm) | ||
+ | |||
+ | |||
+ | :'''3) 3rd day:''' | ||
+ | :- Inoculate 100 µL from the cells culture (grown overnight) in 10 mL of TYM | ||
+ | :- Grow the 10 mL culture until it reach the OD600=0.2-0.6 (60 to 120 min.) | ||
+ | :- Add the entire content of the Erlenmyer (10mL) into another Erlenmyer containing 40 of TYM | ||
+ | :- Grow the culture until it reach the OD600=0.5-0.9 (60 to 120 min.) | ||
+ | :- Add the entire content of the Erlenmyer (50mL) into another Erlenmyer containing 200 of TYM | ||
+ | :- Grow the culture until it reach the OD600=0.6 (90 to 150 min.) | ||
+ | :- Grow the culture until it reach the OD600=0.6 (90 to 150 min.) | ||
+ | :- Cool down quickly (ice bath) until it reach the desired OD! | ||
+ | :1. Centrifuge the cells at 4000 rpm (in 50 mL Falcon tubes) during 15 minutes at 4°C. | ||
+ | :2. Discard the supernatant, resuspend the cell pellet GENTLY in 100mL ice cold TFB1 (initiate with 2mL for each tube, pass through only 2 tubes and complete each tube for 50mL ) | ||
+ | :3. Centrifuge for 8 minutes at 4°C (4000 rpm) | ||
+ | :4. Resuspend GENTLY in 10mL ice cold TFB2 (initiate with 2mL for each tube, put it all together in one tube only and complete the volume to 10 mL with TFB2) | ||
+ | :5. Aliquot 100µl/Eppendorf tube (COLD, new and sterile) | ||
+ | :: Flash freeze in liquid nitrogen and store in the -80°C freezer | ||
Revision as of 15:37, 27 September 2011
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Contents |
Preparation of Chemically competent E. coli cells
A) Lab materials and solutions
- 1) TYM (for 1 L)
- Tryptone - 20 g
- Yeast extract - 5 g
- NaCl - 5.8 g
- MgSO4.7H2O - 2.5 g
- AUTOCLAVE
- Prepare more than 1 Erlenmeyer for each volume (for security reasons)
- 2) TFB1 and TFB2 (storage under refrigeration)
TFB1 for 1L for 200 mL KOAC (Potassium acetate) - 0.03M - 2.94 g → 0.59 g MnCl2.4H2O - 0.05M - 9.90 g → 1.98 g KCl - 0.10M - 7.46 g → 1.49 g CaCl2.2H2O - 0.01M - 1.47 g → 0.29 g Glycerol (glycerin) - 15%(m/V) - 150 mL → 30 mL
- 100 ml of this solution will be used in the procedure
- Filtration required (0.45µm filters) – In the flux! (for sterilization)
TFB2 for 1L for 200 mL MOPS - 0.01M - 2.04 g → 0.41 g KCl - 0.01M - 0.75 g → 0.15 g CaCl2.2H2O - 0.075M - 11.03 g → 2.21 g Glycerol (glycerin) - 15%(m/V) - 150 mL → 30 mL
- Adjust the pH to 7 with KOH
- Filtration required (0.45µm filters) – In the flux! (for sterilization)
- 3) 1 culture plate with solid LB medium WITHOUT ANTIBIOTIC !
- To grow the colonies from the stock of competent cells
B) Material to be checked or prepared in advance
- 1- Prepare the solutions / Place Eppendorf and Falcon in the freezer
- 2- Make reservation for the following equipments:
- - Incubator 37°C (for colonies growth in the culture plate)
- - Shaker 37°C (for cells growth)
- - Spectrophotometer (measures at OD600)
- - Table centrifuge (for 50 mL Falcon)
- - Laminar Flux hood
C) Proceedings
- 1) 1st day: Inoculate cells in the with solid LB medium without antibiotic (37°C) –overnight (inoculate by the end of the afternoon)
- 2) 2nd day: Select colonies and let them grow overnight in a 40 mL Erlenmyer with TYM (shaker 37°C, 200 rpm)
- 3) 3rd day:
- - Inoculate 100 µL from the cells culture (grown overnight) in 10 mL of TYM
- - Grow the 10 mL culture until it reach the OD600=0.2-0.6 (60 to 120 min.)
- - Add the entire content of the Erlenmyer (10mL) into another Erlenmyer containing 40 of TYM
- - Grow the culture until it reach the OD600=0.5-0.9 (60 to 120 min.)
- - Add the entire content of the Erlenmyer (50mL) into another Erlenmyer containing 200 of TYM
- - Grow the culture until it reach the OD600=0.6 (90 to 150 min.)
- - Grow the culture until it reach the OD600=0.6 (90 to 150 min.)
- - Cool down quickly (ice bath) until it reach the desired OD!
- 1. Centrifuge the cells at 4000 rpm (in 50 mL Falcon tubes) during 15 minutes at 4°C.
- 2. Discard the supernatant, resuspend the cell pellet GENTLY in 100mL ice cold TFB1 (initiate with 2mL for each tube, pass through only 2 tubes and complete each tube for 50mL )
- 3. Centrifuge for 8 minutes at 4°C (4000 rpm)
- 4. Resuspend GENTLY in 10mL ice cold TFB2 (initiate with 2mL for each tube, put it all together in one tube only and complete the volume to 10 mL with TFB2)
- 5. Aliquot 100µl/Eppendorf tube (COLD, new and sterile)
- Flash freeze in liquid nitrogen and store in the -80°C freezer