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From 2011.igem.org

October 9th - October 15th

Sunday, October 9

iGEM 2011 Regionals Jamboree: Americas!

Monday, October 10

iGEM 2011 Regionals Jamboree: Americas Award Ceremony

• Cornell brings home the Gold
• Cornell advances to the iGEM 2011 World Jamboree!

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Outreach

• Attended meetings and added input for both AlumniGEM and Community Bricks.

Tuesday, October 11

Wednesday, October 12

Lab work done by: Charlie Chung

Picked Colonies: four (40mL LB + 40µL ampicillin) cultures shaking at 37°C

- AviTagged GFP

- AviTagged VioA from PCR deletion

- AviTagged VioB from PCR deletion

- AviTagged VioE from PCR deletion

Thursday, October 13

Lab work done by: Charlie Chung

Picking Colonies

• (40mL LB + 40µL ampicillin) culture of empty pZE12 vector as a negative control shaking at 37°C

Miscellaneous

• Freshly made 1000x ampicillin and 1M IPTG are in the DNA cryobox of Olin 301 (-20°C fridge)

Subculturing of AviTagged Vio Enzymes

• 20mL from 40mL cultures of VioA, B, E were transferred to (1L LB + 1mL ampicillin) flask for a 1:50 dilution

- Wanted OD to be between 0.05-0.08 after a 2-3hr incubation period, but accidentally let it grow over to 0.1xx

- Regardless, induced with 1mL 1M IPTG

(*EDIT*): Supposed to be a 0.1µM induction with IPTG, so should have added 100µL and not 1000µL to a 1000mL culture. Overinducing may lead to insoluble fractions.

- Shaking at room temp in Olin 301 for 20+ hours until 6pm on Friday (when we'll spin the cells down)

Subculturing of AviTagged GFP

• two 10mL from 40mL culture of GFP were transferred to two (500mL LB + 500µL ampicillin) flasks for a 1:50 dilution

- Wanted OD to be 0.05-0.08 after a 2-3hr incubation period, but accidentally let it grow over to 0.1xx

- Regardless, induced with 500µL 1M IPTG

(*EDIT*): Supposed to be a 0.1µM induction with IPTG, so should have added 50µL and not 500µL to a 500mL culture. Overinducing may lead to insoluble fractions.

- Shaking at room temp in Olin 301 for 20+ hours until 6pm on Friday (when we'll spin the cells down)

Preparing Freezer Stocks

• Took 500µL from remainder of the 40mL cultures and added 500µL of 30% glycerol to create freezer stocks of GFP and VioA, B, E

- In -80°C freezer of Olin 303

Preparation for Sequencing Confirmation of GFP and Vio Colonies

• Miniprep purification of 5mL from remainder of the 40mL cultures. Eluted products are in -20°C fridge. To be submitted for sequencing

Friday, October 14

Lab work done by: Charlie Chung

Subculturing of Negative Control (empty pZE12 vector)

• Transfer two 10mL samples from 40mL culture of empty pZE12 vector to two (500mL LB + 500µL ampicillin) flasks for a 1:50 dilution
• Incubate overnight on 37°C shaker in Olin 304

Protein Extraction of GFP and VioA, B, E

• Spin down 1L cultures into a pellet (10 min at 3200rpm)
• Resuspend in 20mL autoclaved water
• Transfer to 50mL conical tube and spin for 10 min at 3200rpm
• Store in -80°C freezer

Western Blot Preparation

• Centrifuge 1.5mL of GFP and VioA, B, E cultures for 10 min at 14,000rpm
• BugBuster will be used for protein extraction

Saturday, October 15

Afternoon lab work done by: Charlie Chung

Preparation of Starter Cultures for pZE12 Vector and Vio Enzymes

• Used freezer stocks to inoculate (5mL LB + 5µL ampicillin) cultures of empty pZE12 vector and VioA, B, E
• Shaking in 37°C incubator of Olin 304

Lysate Extraction of E. coli with Empty pZE12 Vector -- Negative Control

• Spin down 1L culture into pellets (10 min at 3200rpm; next time do longer and faster spin)
• Stored in -20°C fridge of Olin 303