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Team:Cornell/Week 21
From 2011.igem.org
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:*Gel electrophoresis of PCR product confirms amplification of GFP-AviTag insert | :*Gel electrophoresis of PCR product confirms amplification of GFP-AviTag insert | ||
:*Gel extraction -- 83.1ng/µL of GFP-AviTag insert | :*Gel extraction -- 83.1ng/µL of GFP-AviTag insert | ||
| - | :'''Digestion Setup of pSB1C3 iGEM Backbone from Well 3A''' | + | :*'''<u>Digestion Setup of pSB1C3 iGEM Backbone from Well 3A</u>''' |
::39.7µL ddH2O | ::39.7µL ddH2O | ||
::5µL 10x NEBuffer 3 | ::5µL 10x NEBuffer 3 | ||
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::50µL Total | ::50µL Total | ||
| - | :'''Digestion Setup of GFP-AviTag Insert''' | + | :*'''<u>Digestion Setup of GFP-AviTag Insert</u>''' |
::30.5µL ddH2O | ::30.5µL ddH2O | ||
::12µL GFP-AviTag insert | ::12µL GFP-AviTag insert | ||
Revision as of 00:26, 25 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
- 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 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
