Team:Cornell/Week 21

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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

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

Monday, October 24

Afternoon lab work done by: Charlie Chung

  • Gel electrophoresis of PCR product confirms amplification of GFP-AviTag insert
  • Gel extraction -- 83.1ng/µL of GFP-AviTag insert
  • Digestion Setup of pSB1C3 iGEM Backbone from Well 3A
39.7µL ddH2O
5µL 10x NEBuffer 3
2.8µL pSB1C3 backbone
0.5µL 100x BSA
1µL EcoRI
1µL PstI
50µL Total
  • Digestion Setup of GFP-AviTag Insert
30.5µL ddH2O
12µL GFP-AviTag insert
5µL 10x NEBuffer 3
0.5µL 100x BSA
1µL EcoRI
1µL PstI
50µL Total
  • Western Blot Results
  • Used α-biotin 1° antibody with HRP to select for the biotinylated AviTag peptide on GFP and VioA, B, E
- 1 minute, 5 minute, and 50 minute film exposure confirmed expression of GFP, VioA, and VioE
- Control lysate of pZE12 vector without AviTag correctly displays no band
- Band for VioB protein expression not showing

Late afternoon lab work done by: Claire Paduano

Tuesday, October 25

Wednesday, October 26

Thursday, October 27

Friday, October 28

Saturday, October 29