(Based on Inoue et al. (1990), Gene, 96:23-28)
- Grow cells (e.g. DH5 α) on LB agar plate at 37° C overnight.
- Inoculate pre-culture in 250ml SOB in a 1L flask, 18-20°C with vigorous shaking to OD600 ~ 0.4-0.6 (normally it takes 24-36 hrs)
- Place the flask on ice for 10 min.
- Pellet the cells at 4000rpm for 10 min at 4° C.
- Gently resuspend the cell in 80ml ice-cold TB and store on ice for 10 min.
- Spin at 4000rpm for 10 min at 4° C
- Gently resuspend the pellet in 20ml ice-cold TB and 1.4ml DMSO, place on ice for 10 min
- Prepare 50-500µl aliquots, freeze immediately in liquid N2 and store at -80°C.
|SOB||Bacto Tryptone||20 g|
|Bacto Yeast Extract||5 g|
|8,6 mM NaCl||0,5 g|
|2,5 mM KCl||2,5 mL (from 1 M stock)|
|H2O||ad 1000 mL|
|pH = 7.0|
|autoclave and add sterile|
|10 mM MgCl2||5 mL (from 2 M stock)|
|10 mM MgSO4||5 mL (from 2 M stock)|
|TB solution||10mM PIPES||116 mL|
|Adjust pH to 6.7 with KOH|
|Add MnCl2 to 55mM, and adjust to final volume.|
|Sterilize by filtration with 0.45µm filter and at 4° C|