Editing Competent-Cells (section)

== RbCl-MnCl2 Method,&nbsp;10<sup>8</sup> cfu/µg ==

'''Buffer A''' (prepare 50 ml per 500 ml culture)<br/>Final concentrations:<br/>100 mM RbCl2 (12 g/L)<br/>50 mM MnCl2·4H2O (9.89 g/L)<br/>30 mM potassium acetate (2.94 g/L)<br/>10 mM CaCl2·2H2O (1.47 g/L)<br/>15% (v/v) glycerol (150 ml/L)<br/>Adjust pH to 5.8 with dilute acetic acid and filter sterilize<br/>Note: Store at 4 °C, protect from light.

'''Buffer B '''(prepare 25 ml per 500 ml culture)<br/>Final concentrations:<br/>10 mM MOPS (2.09 g/L)<br/>10 mM RbCl2 (1.2 g/L)<br/>75 mM CaCl2·2H2O (11 g/L)<br/>15% (v/v) glycerol (150 ml/L)<br/>Adjust pH to 6.5 with dilute NaOH and filter sterilize

'''Recipe 2'''

'''Buffer A'''<br/>KOAc 30 mM 2.94g<br/>RbCl2 100 mM 12.10g<br/>CaCl2 10 mM 1.47g,&nbsp;&nbsp;<br/>MnCl2 50 mM 9.90g<br/>Hexamine cobalt Cl 3 mM 0.80g

Glycerol 15% 150ml

pH 5.8 (0.2M acetic acid)

Sterilise by filtration

<br/>'''Buffer B'''<br/>Per 200 ml<br/>MOPS (or PIPES) 10 mM 419mg<br/>CaCl2 75 mM 2.205g&nbsp;<br/>RbCl2 10 mM 2.418g<br/>Glycerol 15% 30ml

pH 6.5 (KOH)<br/>Sterilise by filtration

<br/>Note: Store at 4 °C and protect from light.

*Inoculate 500 ml LB broth in a 1 L flask with 500 µl overnight&nbsp;''E. coli&nbsp;''culture and grow at 37 °C (200 RPM) until OD<sub>600</sub>&nbsp;= 0.5 (about 4 h).
*Chill cells on ice for 10 min.
*Centrifuge ''E. coli ''cells at 2,000 ''x g'' for 10 min at 4 °C.
*Remove supernatant and resuspend the pellet in 50 ml ice-cold buffer A. Keep the cells on ice for the entire time and shake manually in circular motions on ice. It will take 20-30 min to resuspend the cells<br/>''Note: Do not pipette the cells up and down, but you may break the pellet with a pipette for faster resuspension.''
*Centrifuge cells at 2,000 ''x g'' for 10 min at 4 °C.
*Remove supernatant and resuspend the pellet in 25 ml ice-cold buffer B by shaking. Keep the cells on ice for the entire time. It will take some time (20-30 min) to resuspend the cells&nbsp;