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From 2010.igem.org

Date: 06/28/2010

• Time:

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 K12 and DH5a, and Putida KT2440)

Procedure: O/N Cell culture

• 1. Put 2ml LB into 15ml falcon 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 falcon 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

• Time: 4:00pm - 5:30pm

Work Done

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

Procedure: Making frozen stock

• 1. Use EtOH and clean the working environment and pipette.
• 2. Get the falcon 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 from the falcon tube.
• 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.

Other: K12, DH5a, and KT2440 frozen stocks are now stored in -80C freezer (Lin LAB)

Date: 06/30/2010

• Time: 10:00am - 1:00pm

Work Done

• A. Ann, Bryce, and I had a short meeting to discuss about purchasing NAs.
• B. Pseudomonas putida KT2440 antibioic tolerence

Procedure: Please refer back to the protocols under Notebook

• The protocol is posted and titled as "Pseudomonas putida KT2440 antibioic tolerence".
• Stock solutions were made in Lin LAB and transported to iGEM LAB space in ERB.
• We placed the falcon tubes with Pseudomonas putida KT2440 + antibiotics in the incubator in ERB (30C).

Note: We checked the temperature of the freezer in one of the labs in ERB and determined that it's -20C (top). Also, we brought some supplies from Lin LAB (falcon tubes & pipette tips) and made a list. The list is posted on the wall of one of the rooms in ERB so we can keep track of things that were taken from Lin LAB.

Other: The antibiotic stocks (Kan & Tet) made are now stored in iGEM box which can be found in -80C freezer (Lin LAB).

Date: 07/01/2010

• Time:

Work Done

Procedure

Note: