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From 2011.igem.org

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

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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

Evening lab work done by: Maneesh Gupta

Microfluidic Chip Construction

- Poured 10:1 ratio of PDMS for microfluidic chip construction

- Placed molds in oven at 60 degrees Celsius overnight

Tuesday, October 25

Afternoon lab work done by: Claire Paduano

Microfluidic Chip Construction

- Cut chips and bonded to glass slide

Wednesday, October 26

Evening lab work done by: Maneesh Gupta

Shear Test

• Prepared 2 coated chips with Atto 590

- Control chip was Atto 590 under no flow

- Test chip was under a flow rate of 5µl/min

- Pictures taken in air every 30min for 2 hours

- Ran overnight for 8.5 hours

Thursday, October 27

Friday, October 28

Saturday, October 29