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Comp Prep

This protocol describes the preparation of competent E. coli cells for transformation. The key steps are: 1) Growing an overnight culture of E. coli, which is then used to inoculate fresh culture medium. 2) Harvesting the cells by centrifugation when the culture reaches an OD600 of 0.45-0.47. 3) Resuspending the cells in solutions containing MgCl2 and CaCl2 to make them competent for DNA uptake. 4) Aliquoting the competent cells into tubes, freezing in dry ice-ethanol, and storing at -80°C.

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102 views3 pages

Comp Prep

This protocol describes the preparation of competent E. coli cells for transformation. The key steps are: 1) Growing an overnight culture of E. coli, which is then used to inoculate fresh culture medium. 2) Harvesting the cells by centrifugation when the culture reaches an OD600 of 0.45-0.47. 3) Resuspending the cells in solutions containing MgCl2 and CaCl2 to make them competent for DNA uptake. 4) Aliquoting the competent cells into tubes, freezing in dry ice-ethanol, and storing at -80°C.

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samamourad
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Adapted from protocol given by Joe Graber

Amber Pollack 7/6/05

PREPARATION OF COMPETENT CELLS (E. coli) The original protocol from Amy uses 1L of E. coli culture. This protocol is scaled down to 100mL, and makes approximately 25 tubes of 200uL aliquots. MgCl26H20 FM = 203.3 203.3g/L = 1M Protocol: or 20.3g/L=0.1M Note: STERILIZE EVERTHING (graduated cylinder and pipettes) 1. Grow E. coli in a 3mL LB broth overnight. 2. Place 500uL of O/N culture into 100mL of LB broth. 3. Monitor growth until density reaches 0.45-0.47 OD600. 4. Quickly immerse flask in ice, and swirl. 5. Spin out cells at 5000g (5.5 x 103 rpm) for 5 minutes. //For 100mL, use 2 sterile centrifuge bottles and pre-cooled GSA rotor in floor Sorvall centrifuge// 6. Resuspend cells in 24mL of ice cold MgCl2 (0.1M). 7. Spin out by running centrifuge up to 4000g (5000rpm). 8. Pour off supernatant. 9. Suspend cells in 24mL of ice cold, sterile CaCl2, 0.1M by gentle pipetting 10. Spin again just up to 4000g (5x103 rpm) 11. Pour off supernatant. 12. Suspend in 4.3mL of 0.1M sterile CaCl2, ice cold. 13. Add 700uL of sterile glycerol, mix well. 14. Dispense 200uL to sterile Eppendorf vials. Snap-freeze in dry ice. 15. Set one microfuge tube aside on ice to test for competency and contamination, transforming a portion of cells with a known concentration of DNA, and a negative control using no DNA.
//Competent cells will uptake the DNA in the first treatment, and plating the recovered culture on selective media will result in many colonies. In the negative control treatment, no DNA should be present and therefore no antibiotic resistance will be conferred to the cellsthere should be no colonies when this treatment is plated on selective media. The negative control does NOT guarantee the batch is uncontaminated and it is recommended a no-DNA control be used for all transformations.//

need 5.08g/250 mL CaCl22H20 FM=147.0g/L = 1M 14.7g/L = 0.1M or 3.67g/250 mL or 1.47g/100 mL

Adapted from protocol given by Joe Graber

Amber Pollack 7/6/05

Method: Preparation of Competent Cells


April 12 1990 Matthew S. Holt

Purpose: To maintain lab stock of highly efficient low background LM 1035 XL1-Blue and SURE competent cells for plasmid/cosmid transformations (Note: not for use with DH5-Alpha cells). Time required: Day 1: Overnight Day 2: Overnight Day 3: 4 hours to grow culture 2 hours to prepare the competent cells Procedure: Day 1 Streak out the E.coli strain on an LBM plate (no ampicillin!) to isolate colonies and incubate at 37 degrees C overnight (16-20 hours). Day 2 Use a sterile inoculating loop to collect cells from a single colony and inoculate 50 ml sterile 1X LBM Grow at 37 degrees C overnight (16-20 hours) in a shaker incubator. Also place 2 flasks of 250 ml 1X LBM in the incubator to equilibrate the temperature of the medium. Day 3 1. Add 25 ml of the overnight culture to each 250 ml LBM flask. Place another flask of 150 ml 1X LBM in the incubator to equilibrate the temperature of the medium. Grow the cultures to OD650 = O.2. (not dense approximately 3 hours). Add 75 ml of equilibrated 1X LBM to each flask and continue incubating for 30 minutes. 2. Pellet the cells in chilled autoclaved large centrifuge bottles using the Beckman J-6 centrifuge and JA 10 rotor (must be cold!) at 5000 rpm for 10 minutes. Subsequent resuspensions may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room. 3. Decant supernatant and resuspend the cells in 1/4 original volume (87.5 ml) ice cold 100 mM MgCl2. Hold on ice for 5 minutes. Transfer the cells to pre-chilled sterile large centrifuge bottles. Spin in the Beckman J- 6 centrifuge for 10 minutes using the JA-20 rotor 4000 rpm at 4 degrees C. 4. Decant the supernatant and resuspend the cells in 1/20 original volume (17.5 ml) of ice cold 100 mM CaCl2. Hold on ice for 20 minutes. Pellet as above 4000 rpm for 10 minutes. 5. Decant the supernatant and resuspend the cell pellet in 1/100 original volume (3.5 ml) of a solution that is 85% v/v 100 mM CaCl2 and 15% v/v glycerol (100%). For each culture processed chill

Adapted from protocol given by Joe Graber Amber Pollack 7/6/05 approximately 15 labeled eppendorf tubes in a dry ice-EtOH bath. Pipet 300 ul cells into each tube and place immediately into the dry ice-EtOH bath. Transfer the frozen competent cell aliquots to -80 degrees C. 6. After the competent cells have been stored for 24 hours check the efficiency of transformation: Use 1 ng 10 ng and 100 ng of any ampicillin resistant plasmid on LBM + Amp plates as per transformation protocol for intact plasmids. Check the background level by plating 50 ul of cells alone on an LBM + Amp plate. Expect yields to be approximately 5x10e7 colonies per ug of supercoiled DNA. Solutions:

100 mM MgCl2:
1:10 dilution of lab stock; sterilize use sterile ingredients or filter

100 mM CaCl2:
1:10 dilution of lab stock; sterilize use sterile ingredients or filter

85% 100 mM CaCl2, 15% glycerol:


42.5 ml 100 mM CaCl2 7.5 ml 100% glycerol 50.0 ml total volume; mix well and use sterile ingredients or filter sterilize

Precautions: Plasmid/cosmid DNA should be considered biohazards and wastes should be disposed of appropriately. References: Sambrook J. Fritsch E.F. and T. Maniatis.(1989). Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press p.1.74.

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