Montclare:Protocol Preparation of Chemically Competent E.coli Cells

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Introduction

This protocol is used to generate stock aliquots of chemically competent E.coli cells for transformation via heat shock. The typical strain used in the lab is the AF-IQ engineered -Phe auxotrophic strain provided by Prof. David Tirrell (Cal. Tech). However, this technique should be applicable to any common E.coli strain. This method was modeled after sections in the article entitled, Generation of Transformation Competent E.coli in Current Protocols in Microbiology. This article, by Nicholas Renzette, was published in 2011 and (supposedly) yields cells with higher transformation efficiency than cells generated from traditional CaCl2-assisted competency due to the RbCl2 substitution. The first documented application of this method in the Montclare Lab was performed on Jan. 18, 2012 by Carlo Yuvienco. The results from plates streaked with 100 µL of transformed cells showed comparable results to that of electrocompetent cells transformed with the same PQE30 plasmid. 10 mL-scale and 100 mL-scale cultures yield approximately 12 and 120, 100 µL aliquots, respectively.

Requirements

Reagents

  1. LB medium
  2. Transformation Buffer I (TB I), 4°C
  3. Transformation Buffer II (TB II), 4°C
  4. Tryptic soy agar (TSA) plates
  5. Dry ice
  6. Regular ice

Equipment

  1. Glass culture tubes
  2. Microcentrifuge tubes
  3. 40 mL round bottom centrifuge tube
  4. Cryocase for microcentrifuge tubes.
  5. -80°C freezer
  6. 37°C shaking incubator

Procedure

Day 0

  1. Prepare TSA plates with appropriate antibiotics, which will provide selection for the particular E.coli strain. If no antibiotic resistance exists, then regular TSA plates will suffice.
  2. Prepare TB I and TB II and store at 4°C overnight.
  3. Prepare LB stock enough for 2 mL starter culture(s) and larger-scale growth (10-100mL)
  4. Autoclave sterilize the following in preparation for Day 1:
    1. LB in culture tubes to sterilize in preparation for starter cultures on Day 1.
    2. LB in either a culture tube for 10 mL-scale culture or 500mL flask for 100 mL-scale culture.
    3. microcentrifuge tubes
    4. 40 mL round bottom centrifuge tube
    5. 200 mL centrifuge bottle, for 100 mL-scale culture

Day 1

  1. Pre-incubate plates at 37°C and thaw cells on ice. After 10 minutes, streak E.coli cells onto a TSA plate with a sterile inoculation loop. Incubate plate at 37°C overnight (~17 hrs).

Day 2

  1. Inoculate 2 mL starter culture of LB, supplemented with appropriate antibiotics (chloramphenicol for AF-IQ cells), with a single colony of E.coli. Grow overnight at 37°C (300-350 rpm) in a shaking incubator.

Day 3

  1. Subculture large-scale culture (10 or 100 mL) of LB, supplemented with appropriate antibiotics (chloramphenicol for AF-IQ cells), with 20 or 200 µL of starter culture. Grow culture at 37°C (300-350 rpm) in a shaking incubator until early log-phase of growth (OD600=0.3+/-0.05). Keep in mind that doubling times for cell density (i.e. OD600) once log-phase is reached is approximately 20 minutes, thus requiring sometimes frequent measurements. Culture times are typically around 2 hrs, but the initial execution of this procedure required 3h45m to obtain an OD600=0.334.

Important note: At this point in the procedure, keeping the cells consistently at 4°C is critical to their transformation efficiency. As such, pre-incubate containers and buffers in ice-water prior to exposure to cells. Limit the exposure of the cells to > 4°C by transporting containers while submersed in the ice-water bath. While resuspending cells in transformation buffers, limit agitation to only a few seconds at a time, immediately returning to ice-water.

  1. Once the OD600=0.3 is achieved, immediately store culture in appropriate centrifuge container and incubate at 4°C for 20 minutes by submersing closed container in ice water bath. While culture is cooling, equip centrifuge with appropriate rotor and pre-chill centrifuge by running at 4°C, 2000 x g for 20 minutes.
  2. Pellet the culture by spinning at 4°C, 2000 x g for 10 minutes.
  3. Ensure that a pellet has indeed collected and decant supernatant. Discard supernatant.
  4. Add 3.3 mL or 33 mL of TB I (pre-chilled) to centrifuge container containing pellet for 10 mL and 100 mL-scale cultures, respectively. Resuspend pellet by gently swirling buffered slurry.
  5. Incubate slurry in ice-water bath for 2 hrs.
  6. With 30 minutes left for ice-water incubation, crush blocks of dry ice to obtain a rough bed of powder. Embed microcentrifuge tubes (1.5 mL or 2 mL) in the bed of powder to pre-chill.
  7. With 20 minutes left for ice-water incubation, pre-chill centrifuge with appropriate rotor as in step 1.
  8. Pellet the slurry by running at 4°C, 2000 x g for 10 minutes.
  9. Decant and discard supernatant.
  10. Resuspend pellet in 1.1 mL or 11 mL of TB II (pre-chilled) for 10 mL and 100 mL-scale cultures, respectively.
  11. Aliquot slurry in 100 µL amounts amongst the pre-chilled microcentrifuge tubes. Return tubes to dry ice for rapid freezing of cell slurry aliquots.
  12. Collect and store aliquots in a cryocase (pre-chilled) for storage at -80°C freezer.

Day 4

  1. Warm 1-2 mL of LB to 37°C.
  2. Test transformation efficiency of the cells. Begin by rapidly thawing an aliquot of the cells, gently rolling the tube between your palms.
  3. Add plasmid DNA, containing appropriate antibiotic resistance for selection, to the thawed slurry and incubate on ice for 10 minutes.
  4. Heat shock the cells by placing the microcentrifuge tube in a microcentrifuge incubator, 37°C, 600 rpm for 5 minutes.
  5. Add 1 mL LB (pre-warmed) to tube containing the cells and incubate at 37°C, 600 rpm for 1 hr.
  6. Plate 100 µL of cell slurry onto a TSA plate, supplemented with appropriate antibiotics for selection of transformants. Alternatively, several dilutions of of the cell slurry may also be prepared and plated. This is particularly applicable to ampicillin -resistant strains as the ß-lactamase represent in the cells may metabolize the ampicillin within the plate to allow growth of weakly ampicillin-resistant satellite colonies.

Notes

  • This procedure will take approximately 7-8 hours.
  • Treat the cells very gently when resuspending in buffers to prevent lysis.
  • Cell density should be ~ 1 x 1010 cells/mL.
  • Cells may be stored at -80°C for > 2 years, however expect negligible reductions in transformation efficiency with time.

Reagent Recipes

Luria-Bertani broth (LB broth)

  • 10 g tryptone
  • 5 g yeast extract
  • 10 g NaCl
  • Bring volume to 1000 ml with MilliQ H20
  • Sterilize immediately by autoclaving
  • Store up to 6 months at room temperature

Transformation buffer I (TB-I)

  • 30 ml 1 M potassium acetate (60 mM final)
  • 40 ml 1 M MnCl2 (80 mM final)
  • 50 ml 1 M RbCl (100 mM final)
  • 5 ml 1 M CaCl2 (10 mM final)
  • 75 ml 15% (v/v) glycerol
  • Adjust volume to 500 ml with MilliQ H2O
  • Filter sterilize
  • Store up to 1 year at 4°C

Transformation buffer II (TB-II)

  • 5 ml 1M MOPS, pH 7.0 (10 mM final)
  • 5 ml 1 M RbCl (10 mM final)
  • 50 ml 1 M CaCl2 (100 mM final)
  • 75 ml 15% (v/v) glycerol
  • Adjust volume to 500 ml with MilliQ H2O
  • Filter sterilize
  • Store up to 1 year at 4°C
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