Preparation of E.coli competent cell

Aim

To generate competent E.coli cell (modified Inoue method, 10^7~10^8 cfu/ug DNA).

Materials

① LB-MgCl2 medium

Bacto Trypton 2.5 g

yeast extract 1.25 g

NaCl 1.25 g

1N NaOH 0.38 mL (not necessary)

DW up to 250 mL

↓　autoclave at 120℃, 10 min

add 2.5 mL of 1M MgCl2

② Competent cell buffer (Store at 4℃, several months)

PIPES 0.302 g (final 10 mM)

CaCl2・2H2O 0.221 g (final 15 mM)

KCl 1.864 g (final 250 mM)

stDDW up to 70 mL

↓

adjust pH to 6.7 by 5M KOH

↓

add 1.088 g of MnCl2 (final 55 mM)

↓

mess up to 100 mL with DDW

↓

filtrate into 50 mL tube and keep on ice

Methods

Streak competent cell onto LB plate w or w/o antibiotic required. (DH5a, JM109: No, BL21C+: chrolam)

incubate 37℃ O/N.

single colony is inoculated to 5 mL LB w or w/o antibiotic (check sensitivity and resistance to anitibiotic).

37℃ O/N

inoculate 2ml or 1ml of preculture to 250ml LB-MgCl2 (1L flask).

grow to OD600=0.3~0.6 at 18℃ (DH5a: 27-35h, BL21: 12-20h, JM109: 12-20h )

cool down the flask by on ice and mix well, then keep for 5 min (Process from here should be on ice).

transfer to autoclaved centrifuge tube (ice cold).

3 krpm, 10 min, 4℃

discard sup completely

suspend (gently on ice) in 20 mL ice cold competent cell buffer.

transfer cell suspension to 50 mL tube.

3 krpm, 10 min, 4℃

resuspend (gently on ice) in 20 mL of ice cold competent cell buffer.

add 1.5 mL of DMSO and mix gently on ice.

transfer 100 μL of cell suspension to ice-cold 0.5 mL tube on ice in cold room.

transfer all tubes into liquid N, immediately. (液体窒素を使わないでダイレクトに-80℃にいれてゆっくり凍らせた方がよいという噂を聞く 2026/2/20 Goto)

store at -80℃

Transformation

Saw the competent cell on ice

add DNA

incubate on ice for 20 min

heat shock, 42℃ for 30 sec ~ 1 min.

if high transformation efficiency is required, cells are rescued by SOC or LB w/o anitibiotic for 1 h at 37℃

before plating.

Remarks

y-goto.icon