"
Team:Cornell/Week 21
From 2011.igem.org
(Difference between revisions)
(→Sunday, October 23) |
|||
| Line 8: | Line 8: | ||
</html><br><br> | </html><br><br> | ||
==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''' | ||
| + | ::''<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 | ||
| + | ::*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== | ==Monday, October 24== | ||
Revision as of 19:41, 23 October 2011
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 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
