Team:Cornell/Week 19

From 2011.igem.org

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(Monday, October 10)
(Thursday, October 13)
 
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<br>
<br>
<html>
<html>
-
<font size="5"> <i> September 25th - October 1st </i></font>
+
<font size="5"> <i> October 9th - October 15th </i></font>
</html><br><br>
</html><br><br>
==Sunday, October 9==
==Sunday, October 9==
-
'''iGEM 2011 Regional Jamborees: Americas!'''
+
'''iGEM 2011 Regionals Jamboree: Americas!'''
==Monday, October 10==
==Monday, October 10==
-
'''iGEM 2011 Regional Jamborees: Americas Award Ceremony'''
+
:'''iGEM 2011 Regionals Jamboree: Americas Award Ceremony'''
-
::*Cornell awarded Gold Award
+
::*Cornell brings home the Gold
-
::*Cornell advances to the iGEM 2011 World Jamborees!
+
::*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''': five (40mL LB + 40µL ampicillin) cultures shaking at 37°C
+
:'''Picked Colonies''': four (40mL LB + 40µL ampicillin) cultures shaking at 37°C
::- AviTagged GFP
::- AviTagged GFP
-
::- Dysfunctional RFP colony (negative control)
 
::- AviTagged VioA from PCR deletion
::- AviTagged VioA from PCR deletion
::- AviTagged VioB from PCR deletion
::- AviTagged VioB from PCR deletion
Line 31: Line 30:
==Thursday, October 13==
==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
 +
::::(<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)
 +
 +
:'''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

Results | Protocol | Notebook | Parts Submitted

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