Microbiology

DH10B chimiocompetent cells preparation

Before starting, put centrifugation bottles, Falcon tubes and ddH2O and or 10% Glycerol at 4°C. Also precool the centrifuges.

12) Inoculate a culture from an overnight culture at 1/50. 13) Grow until OD600 ~ 0.5-0.6 14) Cool the culture in an ice/waterbath slurry 15) Spin the bacteria at 4°C for 10 minutes at 3500 rpm. 16) Resuspend gently by swirling in an ice/waterbath slurry in 1/3.125 volume of CCMB80 buffer. 17) Incubate on ice for 20 minutes. 18) Spin the bacteria at 4°C for 10 minutes at 3500 rpm. 19) Resuspend in 1/12.5 volume of CCMB80 buffer. 20) Incubate on ice for 20 minutes. 21) Aliquot the cells by 50 µL (volume required for transformation of standard vectors) or aliquot by 100 µL for BAC transformation.

CCMB80 Buffer: KoAC CaCl2·2H2O MnCl2·4H2O MgCl2·6H2O Glycerol

10 mM pH=7 80 mM 20 mM 10 mM 10%

(10 mL of a 1N stock solution) (11.8 gr.L-1) (4 gr.L-1) (2 gr.L-1) (100 mL.L-1)

Adjust pH DOWN to 6.4 with 0.1N HCl Filter-sterilize and store at 4°C Volumes: Starting 250 mL 100 mL

1st wash 80 mL 32 mL

2nd wash 20 mL 8 mL

Microbiology 5