Team:UNICAMP-EMSE Brazil/protocols/chimiocompetent prepare
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:'''2) TFB1 and TFB2''' (storage under refrigeration) | :'''2) TFB1 and TFB2''' (storage under refrigeration) | ||
- | '''TFB1 | + | ::{|border="1" cellpadding="5" cellspacing="0" align="left" |
- | KOAC (Potassium acetate) | + | |- |
- | MnCl2.4H2O | + | |'''TFB1''' |
- | + | | | |
- | CaCl2.2H2O | + | |'''for 1L''' |
- | Glycerol (glycerin) | + | |'''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 | ||
+ | |} | ||
+ | <br clear="all"> | ||
+ | |||
:*100 ml of this solution will be used in the procedure | :*100 ml of this solution will be used in the procedure | ||
:*Filtration required (0.45µm filters) – In the flux! (for sterilization) | :*Filtration required (0.45µm filters) – In the flux! (for sterilization) | ||
+ | ::{|border="1" cellpadding="5" cellspacing="0" align="left" | ||
+ | |- | ||
+ | |'''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 | ||
+ | |} | ||
+ | <br clear="all"> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
:*Adjust the pH to 7 with KOH | :*Adjust the pH to 7 with KOH | ||
:*Filtration required (0.45µm filters) – In the flux! (for sterilization) | :*Filtration required (0.45µm filters) – In the flux! (for sterilization) |
Latest revision as of 20:27, 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