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Team:Cornell/Week 21
From 2011.igem.org
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:::*Measure absorbance at 595nm using spectrophotometer | :::*Measure absorbance at 595nm using spectrophotometer | ||
:::*Calculate volume of sample to be added for 40µg/mL of total protein | :::*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 | ::*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 | ::*Load 10µL ladder, 11.4µL control lysate, 24.4µL VioA, 13.4µL VioB, 14.4µL VioE, 13.4µL GFP | ||
| Line 33: | Line 33: | ||
::*Wash membrane in TBS for 10 minutes | ::*Wash membrane in TBS for 10 minutes | ||
::*Block with 5% dry milk in 20mL of TBS overnight | ::*Block with 5% dry milk in 20mL of TBS overnight | ||
| - | + | ---- | |
:'''PCR of GFP-AviTag-pZE12 to Construct Prefix-GFP-AviTag-Stop-Suffix''' | :'''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) | ::*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 | + | :::- 2.5µL 10mM '''<u>dNTPs</u>''' |
:::- 51°C and 1 minute for annealing temperature and cycle duration | :::- 51°C and 1 minute for annealing temperature and cycle duration | ||
:::- 1µL of template | :::- 1µL of template | ||
==Monday, October 24== | ==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 | ||
| + | :*'''<u>Digestion Setup of pSB1C3 iGEM Backbone from Well 3A</u>''' | ||
| + | :::39.7µL ddH2O | ||
| + | :::5µL 10x NEBuffer 3 | ||
| + | :::2.8µL pSB1C3 backbone | ||
| + | :::0.5µL 100x BSA | ||
| + | :::1µL EcoRI | ||
| + | :::<u>1µL PstI</u> | ||
| + | :::50µL Total | ||
| + | |||
| + | :*'''<u>Digestion Setup of GFP-AviTag Insert</u>''' | ||
| + | :::30.5µL ddH2O | ||
| + | :::12µL GFP-AviTag insert | ||
| + | :::5µL 10x NEBuffer 3 | ||
| + | :::0.5µL 100x BSA | ||
| + | :::1µL EcoRI | ||
| + | :::<u>1µL PstI</u> | ||
| + | :::50µL Total | ||
| + | |||
| + | :*'''<u>Western Blot Results</u>''' | ||
| + | ::*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== | ==Tuesday, October 25== | ||
| + | Afternoon lab work done by: Claire Paduano | ||
| + | :'''Microfluidic Chip Construction''' | ||
| + | ::- Cut chips and bonded to glass slide | ||
==Wednesday, October 26== | ==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== | ==Thursday, October 27== | ||
Latest revision as of 18:01, 28 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 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
