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Team:Cornell/Week 20
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::*Inoculate two baffled flasks of (50mL LB + 50µL ampicillin) each with 1mL of yesterday's culture for a 1:50 dilution | ::*Inoculate two baffled flasks of (50mL LB + 50µL ampicillin) each with 1mL of yesterday's culture for a 1:50 dilution | ||
::*Shake at 37°C for 2.5 hours and check OD reading (if 0.05-0.08, then ready for IPTG-induction) | ::*Shake at 37°C for 2.5 hours and check OD reading (if 0.05-0.08, then ready for IPTG-induction) | ||
| + | ::*OD after 2.5 hours = 0.1xx (VioA), 0.08 (VioB), 0.1xx (VioE) -- too dense again... | ||
| + | :::- Next time, check OD after 2 hours when doing a 1:50 subculture | ||
| + | ::*Induced with 5µL 1M IPTG for 0.1µM addition to a 50mL culture | ||
| + | ::*Shaking at room temperature for 20+ hours, after which we will pellet the cells for lysis and protein extraction | ||
| + | |||
| + | Night lab work done by: Nicholas Kramer | ||
| + | :'''Making Microfluidic Chips''' | ||
| + | ::*Poured 55g of 10:1 PDMS over the molds and put in 60°C oven overnight | ||
==Monday, October 17== | ==Monday, October 17== | ||
| + | Morning lab work done by: Jim Mathew, Charlie Chung | ||
| + | :*Submitted VioA, B, E and GFP for sequencing to confirm PCR deletion | ||
| + | |||
| + | :'''Preparing VioA, B, E and pZE12 for Protein Extraction''' | ||
| + | ::*Spin down the 100mL of each culture into pellets (3700rpm for 30 min) | ||
| + | :::- Stored in -20°C fridge of Olin 301 | ||
| + | |||
| + | Afternoon lab work done by: Maneesh Gupta and Claire Paduano | ||
| + | :'''Making Microfluidic Chips''' | ||
| + | ::*Cut PDMS off of mold and bonded to glass slide with O2 plasma | ||
| + | ::*Tested resulting chips, 6 new chips made | ||
| + | ::*Poured 55g of 10:1 PDMS on molds and put in the 60°C oven overnight | ||
==Tuesday, October 18== | ==Tuesday, October 18== | ||
| + | Afternoon lab work done by: Bill Jo | ||
| + | :'''Making Microfluidic Chips''' | ||
| + | ::*Cut out PDMS and bonded to glass slide using O2 plasma | ||
| + | ::*Tested resulting chips, 3 new chips made | ||
| + | ::*Poured 55g of 10:1 PDMS over mold and put in the 60°C oven overnight | ||
==Wednesday, October 19== | ==Wednesday, October 19== | ||
| + | Morning lab work done by: Bill Jo | ||
| + | :'''Making Microfluidic Chips''' | ||
| + | ::*Cut out PDMS and bonded to glass slide using O2 plasma | ||
| + | ::*Tested resulting chips, 8 new chips | ||
| + | ---- | ||
| + | Afternoon lab work done by: Jim Mathew, Youjin Cho | ||
| + | :'''Lysing GFP and pZE12 Control''' | ||
| + | ::*Lysed the GFP and pZE12 control using BugBuster | ||
| + | ::*After adding 5mL of BugBuster to each sample, shook in incubator for 20 minutes | ||
| + | ::*Spun the samples down for 20 minutes at 4°C at maximum speed | ||
| + | :::(Lysed samples of GFP and pZE12 control stored in 4°C) | ||
==Thursday, October 20== | ==Thursday, October 20== | ||
==Friday, October 21== | ==Friday, October 21== | ||
| + | Afternoon lab work done by: Archana Rachakonda, Jim Mathew, Charlie Chung | ||
| + | :*Spin down 1L cultures of newly transformed (because of sequencing results negative for PCR deletion) VioA, B, E and pZE12 | ||
| + | ::- Stored in -20°C freezer of Olin 301 | ||
| + | |||
| + | :*PCR of GFP-AviTag-pZE12 to create final construct of Prefix-GFP-AviTag-Stop-Suffix | ||
| + | :*Run on gel | ||
| + | ::- PCR failed: no bands of amplified product | ||
| + | |||
| + | :*Redo PCR with two adjustments | ||
| + | ::(1) Lower annealing temperature from 53.2°C to 50.0°C | ||
| + | ::(2) Increase volume of GFP-AviTag-pZE12 template from 0.5µL to 1µL | ||
==Saturday, October 22== | ==Saturday, October 22== | ||
| + | Lab work done at Olin Hall by: Jim Mathew, Charlie Chung | ||
| + | :*Lysed cell pellets of VioA, B, E and empty pZE12 vector using BugBuster | ||
| + | ::- '''''<u>Note</u>''''': VioB pellet is red. Acting on its substrate somewhere in the culture? Confirms active status of enzyme? | ||
| + | ::- Transferred lysate to teammates in Weill Hall running experiment of binding Vio enzymes to microfluidics chips and passing substrate (L-tryptophan) through | ||
| + | |||
| + | :*Gel electrophoresis on PCR product to verify amplification | ||
| + | ::- PCR with two adjustments still failed: only bands of the ladder showed | ||
| + | |||
| + | Lab work done at Weill Hall by: Maneesh Gupta, Archana Rachakonda, Jim Mathew | ||
| + | :*Ran full test of the Biofactory | ||
| + | ::- 4 groups in parallel with 3 coated chips in each group | ||
| + | ::- Enzymes were incubated in the chips for 1 hour with no flow. The enzyme solution was then replace with fresh enzyme solution and incubated again for 1 hr | ||
| + | ::- Flowed L-tryptophan solution through all 4 groups at 5µl/min for 6 hours | ||
| + | ::- Collected flow-through for analysis | ||
| + | ::*Group 1 | ||
| + | :::VioA chip, VioB chip and VioE chip | ||
| + | ::*Group 2 | ||
| + | :::VioA chip, Blank chip, VioE chip | ||
| + | ::*Group 3 | ||
| + | :::VioA chip, VioB chip, Blank chip | ||
| + | ::*Group 4 | ||
| + | :::Blank chip, Blank chip, Blank chip | ||
Latest revision as of 18:02, 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 16th - October 22nd
Sunday, October 16
Morning lab work done by: Charlie Chung
- Preparing 100mL Cultures of VioA, B, E and pZE12 Vector
- Inoculate two baffled flasks of (50mL LB + 50µL ampicillin) each with 1mL of yesterday's culture for a 1:50 dilution
- Shake at 37°C for 2.5 hours and check OD reading (if 0.05-0.08, then ready for IPTG-induction)
- OD after 2.5 hours = 0.1xx (VioA), 0.08 (VioB), 0.1xx (VioE) -- too dense again...
- - Next time, check OD after 2 hours when doing a 1:50 subculture
- Induced with 5µL 1M IPTG for 0.1µM addition to a 50mL culture
- Shaking at room temperature for 20+ hours, after which we will pellet the cells for lysis and protein extraction
Night lab work done by: Nicholas Kramer
- Making Microfluidic Chips
- Poured 55g of 10:1 PDMS over the molds and put in 60°C oven overnight
Monday, October 17
Morning lab work done by: Jim Mathew, Charlie Chung
- Submitted VioA, B, E and GFP for sequencing to confirm PCR deletion
- Preparing VioA, B, E and pZE12 for Protein Extraction
- Spin down the 100mL of each culture into pellets (3700rpm for 30 min)
- - Stored in -20°C fridge of Olin 301
Afternoon lab work done by: Maneesh Gupta and Claire Paduano
- Making Microfluidic Chips
- Cut PDMS off of mold and bonded to glass slide with O2 plasma
- Tested resulting chips, 6 new chips made
- Poured 55g of 10:1 PDMS on molds and put in the 60°C oven overnight
Tuesday, October 18
Afternoon lab work done by: Bill Jo
- Making Microfluidic Chips
- Cut out PDMS and bonded to glass slide using O2 plasma
- Tested resulting chips, 3 new chips made
- Poured 55g of 10:1 PDMS over mold and put in the 60°C oven overnight
Wednesday, October 19
Morning lab work done by: Bill Jo
- Making Microfluidic Chips
- Cut out PDMS and bonded to glass slide using O2 plasma
- Tested resulting chips, 8 new chips
Afternoon lab work done by: Jim Mathew, Youjin Cho
- Lysing GFP and pZE12 Control
- Lysed the GFP and pZE12 control using BugBuster
- After adding 5mL of BugBuster to each sample, shook in incubator for 20 minutes
- Spun the samples down for 20 minutes at 4°C at maximum speed
- (Lysed samples of GFP and pZE12 control stored in 4°C)
Thursday, October 20
Friday, October 21
Afternoon lab work done by: Archana Rachakonda, Jim Mathew, Charlie Chung
- Spin down 1L cultures of newly transformed (because of sequencing results negative for PCR deletion) VioA, B, E and pZE12
- - Stored in -20°C freezer of Olin 301
- PCR of GFP-AviTag-pZE12 to create final construct of Prefix-GFP-AviTag-Stop-Suffix
- Run on gel
- - PCR failed: no bands of amplified product
- Redo PCR with two adjustments
- (1) Lower annealing temperature from 53.2°C to 50.0°C
- (2) Increase volume of GFP-AviTag-pZE12 template from 0.5µL to 1µL
Saturday, October 22
Lab work done at Olin Hall by: Jim Mathew, Charlie Chung
- Lysed cell pellets of VioA, B, E and empty pZE12 vector using BugBuster
- - Note: VioB pellet is red. Acting on its substrate somewhere in the culture? Confirms active status of enzyme?
- - Transferred lysate to teammates in Weill Hall running experiment of binding Vio enzymes to microfluidics chips and passing substrate (L-tryptophan) through
- Gel electrophoresis on PCR product to verify amplification
- - PCR with two adjustments still failed: only bands of the ladder showed
Lab work done at Weill Hall by: Maneesh Gupta, Archana Rachakonda, Jim Mathew
- Ran full test of the Biofactory
- - 4 groups in parallel with 3 coated chips in each group
- - Enzymes were incubated in the chips for 1 hour with no flow. The enzyme solution was then replace with fresh enzyme solution and incubated again for 1 hr
- - Flowed L-tryptophan solution through all 4 groups at 5µl/min for 6 hours
- - Collected flow-through for analysis
- Group 1
- VioA chip, VioB chip and VioE chip
- Group 2
- VioA chip, Blank chip, VioE chip
- Group 3
- VioA chip, VioB chip, Blank chip
- Group 4
- Blank chip, Blank chip, Blank chip
