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Team:Cornell/Week 19
From 2011.igem.org
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| - | <font size="5"> <i> | + | <font size="5"> <i> October 9th - October 15th </i></font> |
</html><br><br> | </html><br><br> | ||
==Sunday, October 9== | ==Sunday, October 9== | ||
| - | '''iGEM 2011 | + | '''iGEM 2011 Regionals Jamboree: Americas!''' |
==Monday, October 10== | ==Monday, October 10== | ||
| - | :'''iGEM 2011 | + | :'''iGEM 2011 Regionals Jamboree: Americas Award Ceremony''' |
| - | ::*Cornell | + | ::*Cornell brings home the Gold |
| - | ::*Cornell advances to the iGEM 2011 World | + | ::*Cornell advances to the iGEM 2011 World Jamboree! |
---- | ---- | ||
:'''Outreach''' | :'''Outreach''' | ||
| - | ::*Attended meetings for both AlumniGEM and Community Bricks. | + | ::*Attended meetings and added input for both AlumniGEM and Community Bricks. |
==Tuesday, October 11== | ==Tuesday, October 11== | ||
| Line 23: | Line 23: | ||
==Wednesday, October 12== | ==Wednesday, October 12== | ||
Lab work done by: Charlie Chung | Lab work done by: Charlie Chung | ||
| - | : | + | :'''Picked Colonies''': four (40mL LB + 40µL ampicillin) cultures shaking at 37°C |
::- AviTagged GFP | ::- AviTagged GFP | ||
::- AviTagged VioA from PCR deletion | ::- AviTagged VioA from PCR deletion | ||
| Line 31: | Line 31: | ||
==Thursday, October 13== | ==Thursday, October 13== | ||
Lab work done by: Charlie Chung | 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 | ||
| + | ::::(<u>'''*EDIT*'''</u>): 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) | ||
| - | :*Miniprep purification of 5mL from remainder of the 40mL cultures. Eluted products are in -20°C fridge. To be submitted for sequencing | + | :'''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 | ||
| + | ::::(<u>'''*EDIT*'''</u>): 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== | ==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== | ==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 | ||
Latest revision as of 19:07, 16 October 2011
Week 1 | Week 2 - 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20 | Week 21 |
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!
- 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
