Materials

• LB media
  • 1% w/v Peptone
  • 1% w/v NaCl
  • 0.5% w/v Yeast Extract
• TSS Solution
  • 85 ml sterile LB media
  • 10 g PEG-3350 in 5 ml H₂O (10%)
  • 5 ml DMSO (5%)
  • 2 ml of 1M MgCl₂ (Hydrous: 1.017g + 5ml H20. Anhydrous: 0.476g + 5mL H2O)
• KCM Solution
  • 500 ml H₂O
  • 18.64 g KCl (500mM)
  • 8.32 g CaCl₂ (150mM)
• Two 2 L conical flasks
• Four 250ml centrifuge bottles
• High-speed centrifuge

Procedure

• Day 1
  1. Prepare two 500 ml aliquots of LB media in two 2 L conical flasks and autoclave
  2. Autoclave four 250 ml centrifuge bottles
  3. From -80ᵒC stocks, streak plate cells to be made competent on agar plates supplemented with appropriate antibiotics. Note: It is advised best not to re-streak from a competent cell stock.
• Day 2
  1. At the end of the day, pick tree colonies to be grown overnight in three separate aliquots of 10 ml of LB media supplemented with the appropriate antibiotics.
  2. Incubate the cells overnight at 37ᵒC, in a shaking incubator set to 210 RPM.
• Day 3
  1. In the morning inoculate the 500 ml flasks of LB media with 5 ml of the overnight cell culture.
  2. Incubate the cells at 37ᵒC, in a shaking incubator set to 210 RPM. Measure the OD600 every hour until OD600 0.4 - 0.6 is achieved.
  3. While the cells incubate, chill the following:
     • The four 250 ml centrifuge bottles – on ice
     • Labelled PCR tubes (prepare 300 for a 1 L cell culture) – on ice
     • Set the centrifuge to 4ᵒC and leave the rotor inside
     • 50 ml TSS solution
  4. Once a OD600 of 0.4-0.6 is achieved, aliquot the culture into the centrifuge tubes and keep on ice to inhibit further growth.
  5. Pellet the cells in the centrifuge by spinning them at 5000 g for 5 min
  6. Discard the supernatant and resuspend the cells in 50 ml ice-cold TSS
  7. Aliquot 200 µl of cell solution into each PCR tube
  8. Directly store the tubes in a -80ᵒC freezer.

Source:

Chung, C., Niemela, S. and Miller, R. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences, 86(7), pp.2172-2175.

Materials

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Sample	Reagent 1	Reagent 2	Water	Total volume
No.1	3	3	4	10
No.2	4	3	3	10
No.3	5	3	2	10

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Procedure

Source: