Lidstrom:Competent Cell Preparation

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Chemically Competent E. Coli

Notes:

• You need fresh cells.
  • Often people inoculate a few mL of the culture for overnight growth, then use 200 uL to inoculate the ~50 mL of culture they will make competent.
  • Some people in our lab believe you want to start with a fresh plate and don't even use one that is a few days old. Other people (Mila) are mostly concerned about the OD being right at the time of harvest.
• You need to flash freeze the cells at the end of the procedure. You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC.

Amanda/Janet Protocol for Chemically Competent Cells

Supplies needed:

• Plate of E. Coli colonies
• SOB-Mg growth medium
  • "-" is "minus", indicating that there is no added Mg
• Sterilized 500 mL Erlenmeyer flasks
• 50 mL screw-cap polypropylene tubes
• Freezer tubes (1.5 mL)
• SOC medium for recovery after heat shock (LB/TB is fine instead)

Method

1. Pick several colonies off a freshly streaked plate into ~1 mL SOB-Mg growth medium
   1. Grow cells overnight or several hours in media with appropriate antibiotics if available.
   2. Use more inoculum in the next step if cultures weren't grown overnight.
2. Inoculate 50 mL SOB-Mg growth medium with this culture. Use 500 uL of stationary phase culture for 50 mL SOB-Mg medium.
3. Incubate at 275 rpm, 37^oC until OD₆₀₀ is about 0.3, which corresponds to ~ 5*10⁷cells/mL
   1. Higher OD isn't usually a problem for routine work.
4. Collect in sterile 50 mL polypropylene centrifuge tube(s) and chill on ice for 10 minutes
5. Pellet the cells at 750 - 1,000g (2500 rpm) for 14 min at 4^oC. Decant the supernatant and invert tubes to remove excess culture medium.
6. Disperse cells in ~1/3 volume of CCMB by gentle vortexing or rapping of the centrifuge tube.
7. Incubate on ice for 20 minutes
8. Centrifuge at 2500 rpm for 10 min at 4^oC
9. Resuspend cells in CCMB at 1/12 the original culture volume
10. Make aliquots in eppendorf tubes, ideally on ice
11. Flash freeze with liquid nitrogen
12. Store at -80oC to preserve them for many months

SOB-Mg growth medium (1 Liter)

Setting cell widths

Ingredient	Amount
Bacto Tryptone	20g
Bacto Yeast Extract	5g
1M NaCl	10mL
1M KCl	2.5mL

• Add water to make 1L
• Autoclave
• Dispense into smaller bottles for lower contamination risk

CCMB (1 Liter)

Setting cell widths

Ingredient	Amount	Final Concentration
Potassium Acetate, 1M, pH 7	10 mL	10mM
Glycerol	100g	10% (w/v)
CaCl2.2H2O	11.8g	80mM
MnCl2.4H2O	4g	20mM
MgCl2.6H2O	2.5mL	10mM

1. Prepare a 1M solution of potassium acetate, pH 7.0 using KOH.
   1. Filter through a 0.2 uM membrane & store frozen
2. Prepare a solution of 10% potassium acetate, 10% glycerol
3. Add salts, allowing each to enter solution before adding the next.
4. Adjust pH to 6.4 with 0.1M HCl. Do not adjust pH upward with base.
5. Filter through a 0.2 uM filter & store at 4^oC.

Nicole/Andrew protocol for Chemically Competent cells

Materials and reagents

• E. coli line (Top 10, S17-1, BL21-AL, BL21-D3, JM109, Qiagen)
• TFB I (transformation buffer)
• TFB II

TFB I (100 ml)

30 mM acetate K (0.294 g)

100 mM RbCl (1.21 g)

10 mM CaCl2 (0.14 g)

50 mM MnCl2 (1.0 g)

15% glycerol (15 ml)

dH2O

pH = 5.8 (use acetic acid to adjust)

TFB II 100 ml

10 mM MOPS (0.21 g)

75 mM CaCl2 (1.1 g)

10 mM RbCl (0.12 g)

15% glycerol (15 ml)

dH2O

pH = 6.5 (use KOH to adjust)

Protocol

• 2 days before making cells, streak out the line of E. coli to make on LB plates (+strep for Top 10 and S17-1)
• 1 day before:

inoculate 4 white capped test tubes or disposable 14 ml clear-top falcon tubes with 1 ml of LB (+strep)

freeze appropriate color, autoclaved epi tubes in -80°C (80+ tubes)

- white tube = Top 10

- yellow tube = S17-1

- pink tube = BL21-AL

- purple tube = BL21-D3

- green tube = JM109

- blue tube = Qiagen

1. Grow cells in 5 ml LB (+5 ul strep for Top 10 and S17-1 cells) overnight
2. Transfer 1 ml of cells to 50 ml LB (use falcon tube) and grow at 37°C for 90 min

   - want OD of 0.4 or 0.5 before starting next steps

3. Place on ice (0°C) for 1 min
4. Spin at 6000g, 0°C for 5 min
5. Add 15 ml cold dH2O
6. Spin at 6000g, 0°C for 5 min, pour off super
7. Add 10 ml cold TFB I to pellet
8. Incubate on ice for 15 min
9. Spin at 6000g, 0°C for 5 min, pour off super
10. Add 1 ml cold TFB II to pellet
11. Incubate on ice for 30 min
12. Aliquot 50 ul into -80°C epi tubes (or into tubes sitting in dry ice)
13. Immediately store at -80°C

• • • Best method

      take tubes out of freezer

      open all caps

      pipette 50 ul into each

      close caps

      back in -80°C

      VERY QUICKLY!

TEST CELLS BEFORE STOCKING FOR GENERAL USE

• For contamination

1. Scrape a sample from frozen stock
2. Streak on LB (no abx)
3. Grow at 37°C overnight
4. Check for contamination (E. coli should be translucent and yellowish) – If none is present test competency

• For competency

Use PCM184 plasmid stock (and Amp or Kan/Tet)

Follow protocol for transformation

Electrocompetent Cells

• You can easily make your own electro-competent cells for electroporation.

1. Grow a small (~ 2 mL) overnight culture with appropriate antibiotic(s)
2. Use overnight to inoculate: generally use ~ 200 uL/50 mL
3. Let grow for about 3-4 h (OD 0.4-0.6 or so)
   1. If using the tube spec, the path length is longer than in the cuvettes. Divide tube specs by 1.65 to convert to the 1 cm path length OD. (If using tube spec, let the OD get to ~1.)
4. Centrifuge and wash 2-3 times with 10% glycerol (everything on ice).
   1. Once or twice is fine. Don't waste time/energy doing much more. -JM 8/2013
5. Remove pretty much all of the supernatant after the last centrifugation
   1. Leave just enough water to allow for pipetting. The volume of supernatant left should be < 1/500^ths of the culture volume.
   2. See Janet's graph below. The really concentrated cells performed well despite having clumped into a serious "booger" during recovery (pre-plating) that was mostly unspreadable.

      

      electroporation: number of colonies versus competent cell density

      1. Undiluted cells in this experiment are actually quite dilute. Glycerol water of volume 1/500th of the culture volume was mixed back in. Janet has subsequently (8/2013) learned that more concentrated is much better, and is now routinely getting lawns from gibson assembly transformations.
6. Aliquot into 1.5 ml centrifuge tubes (40-50uL in each), then flash freeze with liquid nitrogen. Store at -80C.

• Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See this page.
• [[User:Janet B. Matsen|Janet's] cells clump into a "booger" during recovery (pre-plating) that was mostly unspreadable. The clumping is less of an issue if you don't centrifuge them before plating. For this reason I recover in 200 uL and plate all of it.