Making Competent cell (Rubidium chloride method)

Making Competent Cells:

• Grow 50mL overnight culture
• Inoculate 1L/500mL LB with 20mL/10mL of the O/N culture
• Grow with shaking at RT to an OD600=0.2 – 0.3 (~7-8 hrs)o Doubling time is every 30 min
• Centrifuge at 3500 rpm in a sterile bottle
• Discard supernatant
• Resuspend gently in 200mL/100mL TB buffer. DO NOT VORTEX
• Incubate on ice for ~20 mins and mix occasionally
• Centrifuge (5 min @ 3500rpm)
• Discard supernatant
• Resuspend in 50mL/25mL TB
• Incubate on ice for ~30 mins and mix occasionally
• Add 1.8mL/0.9mL DMSO and invert to mix
• Aliquot into cold tubes
• Freeze in liquid N2 and store at -80°CTB BUFFER [make fresh buffer each time] Filter Sterilize and keep on ice