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Team:Cornell/Week 21
From 2011.igem.org
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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
- Bradford Assay to Determine Protein Concentration of Samples
- 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
- PCR of GFP-AviTag-pZE12 to Construct Prefix-GFP-AviTag-Stop-Suffix
- Previous failures of PCR were due to accidental use of dATP instead of dNTPs (label on Eppendorf was messy and ambiguous)
- - 2.5µL 10mM dNTPs
- - 51°C and 1 minute for annealing temperature and cycle duration
- - 1µL of template
