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From 2010.igem.org

Date: 06/28/2010

Work Done

• A. Ann, Bryce, and I had a short meeting with Mike (Graduate Student Advisor) to discuss about HPLC column.
• B. O/N Cell culture (E. Coli / DH5A )

Procedure: O/N Cell culture

• 1. Put 2ml LB into 15ml test tube (for incubation).
• 2. Get a stock of DH5a stored in -80C freezer. (Ann: K12, Bryce: KT2440)
• 3. Use a pipette tip and scratch the surface of the stock, then put the pipette tip into the 15ml test tube.
• 4. Place the test tube into 30C incubator for O/N.

Note: In order to carry out the procedure in sterile environment, clean the lab table with EtOH and turn on the flame. Make sure to work near the flame and sterilize the test tube opening and its cap with the flame quickly. When transfering the stock, use a new pair of gloves. In addition, -80 freezer must not be opened for more than 20 seconds.

Date: 06/29/2010

Word Done

• A. Ann, Bryce, and I had a short meeting in Duderstadt to discuss about future plan.
• B. Made a forzen stocks for K12, DH5a, and KT2440 that we incubated the day before.

Procedure: Making frozen stock

• 1. Use EtOH and clean the working environment and pipette.
• 2. Get the test tubes with cell culture from the 30C incubator and have the flame on.
• 3. Take 1.5ml test tube and transfer 1ml of the cell cultures.
• 4. Centrifuge the tube: 13000 RPM for 1 minute.
• 5. Remove the liquid out without disturbing the pellet at the bottom.
• 6. Resuspend the pellet with 0.5ml of fresh LB.
• 7. Add 0.5ml of 50% glycerol.
• 8. Transfer the mixture into a Cryotube.
• 9. Place the cryotube into -80C freezer.

Note: Be careful handling the test tubes. Sterilize their openings with flame quickly and avoid any possible contamination. The centrifuge must be balanced. After the spin, Cell pellet can be dettached so handle the tubes with extra caution. Put LB first and glycerol second because glycerol is very viscous and suspending the cells can be difficult.

Date: 06/30/2010