Team:Cornell/Week 21

From 2011.igem.org

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(Sunday, October 23)
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==Sunday, October 23==
==Sunday, October 23==
 +
Morning lab work done by: Charlie Chung
 +
:'''Western Blot to Confirm Expression of VioA, B, E and GFP in Cell Lysate'''
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::''<u>Bradford Assay to Determine Protein Concentration of Samples</u>''
 +
:::*Prepare 6 serial dilutions of BSA (10mg/mL from NEB) in triplicate
 +
::::- Add 18.4µL ddH2O to wells A1, B1, C
 +
::::- Add 1.6µL BSA to Column 1
 +
::::- Add 10µL ddH2O to wells A2-7, B2-7, C2-7
 +
::::- Mix and pipet up 10µL from Column 1. Transfer to Column 2 and mix. Discard tips
 +
::::- Repeat down the columns. For Column 7, discard 10µL after mixing
 +
 +
:::*Prepare dilution of control lysate; VioA, B, E; and GFP samples
 +
::::- Add 8µL ddH2O to wells A8-12, B8-12, C8-12
 +
::::- Add 2µL of control lysate to Column 8; VioA to Column 9; VioB to Column 10; VioE to Column 11; GFP to Column 12
 +
 +
:::*Add 100µL 1X Bio-Rad Protein Assay Bradford Dye to all wells (both BSA standard and samples)
 +
:::*Wait 10 minutes
 +
:::*Measure absorbance at 595nm using spectrophotometer
 +
:::*Calculate volume of sample to be added for 40µg/mL of total protein
 +
::*Add 7µL of protein loading dye (with β-mercaptoethanol) to 35µL sample and boil for 10 minutes at 95°C
 +
::*Load 10µL ladder, 11.4µL control lysate, 24.4µL VioA, 13.4µL VioB, 14.4µL VioE, 13.4µL GFP
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::*Run for 1 hour at 90V
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::*Transfer onto PVDF membrane for 1 hour at 80mAmp
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::*Wash membrane in TBS for 10 minutes
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::*Block with 5% dry milk in 20mL of TBS overnight
==Monday, October 24==
==Monday, October 24==

Revision as of 19:41, 23 October 2011

Results | Protocol | Notebook | Parts Submitted

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October 23rd - October 29th

Sunday, October 23

Morning lab work done by: Charlie Chung

Western Blot to Confirm Expression of VioA, B, E and GFP in Cell Lysate
Bradford Assay to Determine Protein Concentration of Samples
  • Prepare 6 serial dilutions of BSA (10mg/mL from NEB) in triplicate
- Add 18.4µL ddH2O to wells A1, B1, C
- Add 1.6µL BSA to Column 1
- Add 10µL ddH2O to wells A2-7, B2-7, C2-7
- Mix and pipet up 10µL from Column 1. Transfer to Column 2 and mix. Discard tips
- Repeat down the columns. For Column 7, discard 10µL after mixing
  • Prepare dilution of control lysate; VioA, B, E; and GFP samples
- Add 8µL ddH2O to wells A8-12, B8-12, C8-12
- Add 2µL of control lysate to Column 8; VioA to Column 9; VioB to Column 10; VioE to Column 11; GFP to Column 12
  • Add 100µL 1X Bio-Rad Protein Assay Bradford Dye to all wells (both BSA standard and samples)
  • Wait 10 minutes
  • Measure absorbance at 595nm using spectrophotometer
  • Calculate volume of sample to be added for 40µg/mL of total protein
  • Add 7µL of protein loading dye (with β-mercaptoethanol) to 35µL sample and boil for 10 minutes at 95°C
  • Load 10µL ladder, 11.4µL control lysate, 24.4µL VioA, 13.4µL VioB, 14.4µL VioE, 13.4µL GFP
  • Run for 1 hour at 90V
  • Transfer onto PVDF membrane for 1 hour at 80mAmp
  • Wash membrane in TBS for 10 minutes
  • Block with 5% dry milk in 20mL of TBS overnight

Monday, October 24

Tuesday, October 25

Wednesday, October 26

Thursday, October 27

Friday, October 28

Saturday, October 29