Team:Rice University/Electrocompetent

For more information visit www.bio-rad.com and search "electroprotocols manual." The purpose of this procedure is to remove as much salt from the media as possible while maintaining cell viability. Additionally, we'll be concentrating the cells cultures to roughly 500x the original titer.

WHAT YOU WILL NEED: large ice bucket O/N cultures of appropriate strains Sterile LB Media - amount depends on how much you would like to make (500mL=50 competent stocks) Sterile 10% Glycerol - approximately twice the culture volume you are going to use. Chill in ice bucket. Sterile 2.0mL microcentrifuge tubes - labeled with strain info Several serological pippettes & bulb

1. Innoculate LB cultures with approximately 1/100 volume of O/N and culture while shaking @ 37 until OD600nm is between 0.6 and 1.0.

2. Pellet cells by spinning @ 4000xg for 10 minutes while chilling @ 4*C. (Cells should be kept as close to 4*C as possible for the remainder of the protocol).

3. Gently pour off supernatant, but try to get most of it off. It is better to lose some cells at this point than keep the LB around. Resuspend the pellet in equal volume of sterile 10% glycerol.

4. Repeat 2 & 3, resuspending in 1/2 the original culture volume.

5. Repeat 2 & 3, resuspending in 1/50 original culture volume.

6. Repeat 2 & 3, resuspending in 1/500 original culture volume.

7. Pippette 40uL aliquots into labeled 2.0mL microcentrifuge tubes and place in -80*C freezer.