Team:Cornell/Week 20

From 2011.igem.org

(Difference between revisions)
(→Wednesday, October 19)
(→Saturday, October 22)
 
(14 intermediate revisions not shown)
Line 47: Line 47:
::*Cut out PDMS and bonded to glass slide using O2 plasma
::*Cut out PDMS and bonded to glass slide using O2 plasma
::*Tested resulting chips, 8 new chips
::*Tested resulting chips, 8 new chips
-
Afternoon lab work done by: James Mathew, Charlie Chung, Youjin Cho
+
----
-
:'''Lysing GFP and pZE12 control'''
+
Afternoon lab work done by: Jim Mathew, Youjin Cho
-
::*Lysed the GFP and pZE12 control using BugBuster.
+
:'''Lysing GFP and pZE12 Control'''
-
::*After adding 5ml of BugBuster to each samples, shook the samples for 20 minutes.
+
::*Lysed the GFP and pZE12 control using BugBuster
-
::*Spun the samples down for 20 minutes at 4°C at maximum speed.
+
::*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)
:::(Lysed samples of GFP and pZE12 control stored in 4°C)
Line 61: Line 62:
::- Stored in -20°C freezer of Olin 301
::- Stored in -20°C freezer of Olin 301
-
:*PCR of GFP-AviTag-pZE12 to create final construct of iGEM prefix-GFP-AviTag-iGEM suffix
+
:*PCR of GFP-AviTag-pZE12 to create final construct of Prefix-GFP-AviTag-Stop-Suffix
-
:*Run on gel, extract, and purify
+
:*Run on gel
-
:*Digest with EcoRI and PstI
+
::- 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

Results | Protocol | Notebook | Parts Submitted

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
Retrieved from "http://2011.igem.org/Team:Cornell/Week_20"