Lidstrom:Competent Cell Preparation

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Revision as of 14:08, 24 August 2012

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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.

Electrocompetent Cells

• You can make your own electro-competent cells for electroporation.
  • From Amada's past mentor in undergrad:
    • Grow the cells overnight
    • Inoculate from this the next morning: generally use 200 uL/50 mL
    • Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 3 times with 10% glycerol (everything on ice).
    • Resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and store at -80C.
      • Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See this page. Would allow you to calculate efficiency.

Nicole/Andrew protocol

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 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