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Team:Cornell/Week 9
From 2011.igem.org
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::*Tested chips to check for blockages in flow | ::*Tested chips to check for blockages in flow | ||
:::Three working devices, one with complete blockage in the channel | :::Three working devices, one with complete blockage in the channel | ||
| + | ::Used protocol provided by Dr Archer from BME lab class | ||
::*Poured more PDMS and baked overnight at 60 degC | ::*Poured more PDMS and baked overnight at 60 degC | ||
Revision as of 15:18, 5 August 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 |
July 31st - August 6th
Sunday
Monday
Tuesday
Microfluidics work done by: Jim, Nick, Dan, Claire, Youjin, Bill
- Microfluidics
- Dr Archer showed us how to use the clean room.
- Made a SU-8 master of our device.
- Poured PDMS and baked overnight at 60 degC
- Used protocol provided by Dr. Archer from BME lab class
Wednesday
Microfluidics work done by: Jim, Nick, Dan, Claire, Youjin, Bill
- Microfluidics
- Dr Archer showed us how to cut out PDMS device and seal to clean glass slides using plasma cleaner.
- Tested chips to check for blockages in flow
- Three working devices, one with complete blockage in the channel
- Used protocol provided by Dr Archer from BME lab class
- Poured more PDMS and baked overnight at 60 degC
Thursday, August 4
Morning lab work done by: Jim Mathew
- Objective
- Prepare Avi-Tagged pZE12 vector backbone for GFP, RFP, and vioE gene inserts
- Digestion Setup in Duplicate
- 32.6μL H2O
- 9.9μL Avi-Tagged pZE12 vector backbone (2μg)
- 5μL 10x NEBuffer 4
- 0.5μL 100x BSA
- 1μL KpnI-HF (HF = high fidelity)
- 1μL SphI-HF
- 50μL Total
- Incubate in 37°C water bath for 2 hours
- Dephosphorylation of 5' Ends of Vector Backbone
- Add 1μL of Calf Intestine Alkaline Phosphatase (CIAP) to digested vector backbone in order to prevent self-ligation without gene insert included
- Incubate at 50°C for 5 minutes
- Run vector backbone through agarose gel electrophoresis
- Gel Extraction and Purification of Digested Backbone
- Followed standard Qiagen Gel Extraction protocol for pZE12 vector backbone that is now Avi-Tagged and digested with KpnI and SphI
- NanoDrop spectrophotometry on the duplicate samples reported 12.7ng/μL and 14.2ng/μL
Afternoon lab work done by: Claire Paduano, Charlie Chung
- Objective
- Ligation of GFP, RFP, and vioE genes with Avi-Tagged pZE12 backbone
- Ligation
- GFP + Avi-Tagged Backbone
- 10.6μL Avi-Tagged pZE12 vector backbone
- 3.6μL GFP
- 2.8μL H2O
- 2.0μL T4 DNA Ligase Buffer
- 1.0μL T4 DNA Ligase
- 20μL Total
- RFP + Avi-Tagged Backbone
- 10.3μL Avi-Tagged pZE12 vector backbone
- 6.7μL RFP
- 2.0μL T4 DNA Ligase Buffer
- 1.0μL T4 DNA Ligase
- 20μL Total
- vioE + Avi-Tagged Backbone
- 10.6μL Avi-Tagged pZE12 vector backbone
- 4.1μL vioE
- 2.3μL H2O
- 2.0μL T4 DNA Ligase Buffer
- 1.0μL T4 DNA Ligase
- 20μL Total
- In all above reactions, volume of plasmid vector added corresponds with 100ng backbone.
- Prepare control ligation reactions (inserts replaced with H2O) for each of the above constructs.
- Same volume of vector backbone, buffer, ligase
- Volumes of all inserts (i.e. gene) go toward volume of H2O
- After ligation setup, incubate in 16°C waterbath overnight.
- Ligation reaction volumes were calculated using a formulated spreadsheet (see Protocol page under the Multimedia tab) that is based on 100ng of vector backbone and a 3:1 molar ratio of insert:vector.
- Reference for Inserts and Vector Backbone
- RFP gene - 678bp
- GFP gene - 717bp
- vioE gene - 576bp
- pZE12 vector - 2340bp
