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Team:Cornell/Week 20
From 2011.igem.org
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Olin lab work done by: Jim Mathew, Charlie Chung | Olin lab work done by: Jim Mathew, Charlie Chung | ||
:*Lysed cell pellets of VioA, B, E and empty pZE12 vector using BugBuster | :*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 enzymes to microfluidics chip and passing substrate (L-tryptophan) through | ::- Transferred lysate to teammates in Weill Hall running experiment of binding enzymes to microfluidics chip and passing substrate (L-tryptophan) through | ||
:*Gel electrophoresis on PCR product to verify amplification | :*Gel electrophoresis on PCR product to verify amplification | ||
::- PCR with two adjustments still failed: only bands of the ladder showed | ::- PCR with two adjustments still failed: only bands of the ladder showed | ||
Revision as of 00:04, 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 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
Olin lab work done 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 enzymes to microfluidics chip 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
