Team:Cornell/Week 9

From 2011.igem.org

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:#Desalted overnight ligation
:#Desalted overnight ligation
:#Transformed the samples into DH5α electrocompetent cells
:#Transformed the samples into DH5α electrocompetent cells
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:'''Group Meeting'''
:'''Group Meeting'''
::*Just finished transforming GFP/RFP/VioE + AviTag + pZE12 backbone
::*Just finished transforming GFP/RFP/VioE + AviTag + pZE12 backbone
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==Saturday, August 6==
==Saturday, August 6==
Lab work done by: Youjin Cho, Charlie Chung
Lab work done by: Youjin Cho, Charlie Chung
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*Miniprepped 5mL cultures of transformed DH5α electrocompetent bacteria (standard Qiagen Miniprep protocol)
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:*Miniprepped 5mL cultures of transformed DH5α electrocompetent bacteria (standard Qiagen Miniprep protocol)
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:NOTE: White masses were floating in bacteria culture -- potential contamination?
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::<u>NOTE</u>: White masses were floating in bacteria culture -- potential contamination?
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*NanoDrop spectrophotometry to determine DNA concentration of 50µL elution product
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:*NanoDrop spectrophotometry to determine DNA concentration of 50µL elution product
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::RFP (Colony 1) + Avi-Tagged pZE12 vector backbone = 53.2ng/µL
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::RFP (Colony 1) + AviTagged pZE12 vector backbone = 53.2ng/µL
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::RFP (Colony 2) + Avi-Tagged pZE12 vector backbone = 63.4ng/µL
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::RFP (Colony 2) + AviTagged pZE12 vector backbone = 63.4ng/µL
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::RFP (Colony 3) + Avi-Tagged pZE12 vector backbone = 76.6ng/µL
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::RFP (Colony 3) + AviTagged pZE12 vector backbone = 76.6ng/µL
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::GFP (Colony 1) + Avi-Tagged pZE12 vector backbone = 65.7ng/µL
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::GFP (Colony 1) + AviTagged pZE12 vector backbone = 65.7ng/µL
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::GFP (Colony 2) + Avi-Tagged pZE12 vector backbone = 65.5ng/µL
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::GFP (Colony 2) + AviTagged pZE12 vector backbone = 65.5ng/µL
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::GFP (Colony 3) + Avi-Tagged pZE12 vector backbone = 46.6ng/µL
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::GFP (Colony 3) + AviTagged pZE12 vector backbone = 46.6ng/µL
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::vioE (Colony 1) did not grow in its culture tube
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::VioE (Colony 1) did not grow in its culture tube
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::vioE (Colony 2) + Avi-Tagged pZE12 vector backbone = 53.8ng/µL
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::VioE (Colony 2) + AviTagged pZE12 vector backbone = 53.8ng/µL
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::DNA from vioE (Colony 3) was accidentally discarded...Charlie is extremely ashamed.
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::DNA from VioE (Colony 3) was accidentally discarded...Charlie is extremely ashamed.

Latest revision as of 17:23, 18 September 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 |


July 31st - August 6th

Sunday, July 31

Monday, August 1

Lab work done by: Jim Mathew

  • Sent VioE and AviTag + backbone in for sequencing

Tuesday, August 2

Microfluidics work done by: Jim Mathew, Nick Kramer, Dan Levine, Claire Paduano, Youjin Cho, Bill Jo

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°C
  • Followed protocol that is used by Biomedical Engineering lab class and is provided by Dr. Archer

Wednesday, August 3

Microfluidics work done by: Jim Mathew, Nick Kramer, Dan Levine, Claire Paduano, Youjin Cho, Bill Jo

Microfluidics
  • Dr. Archer showed us how to cut out PDMS devices and seal to clean glass slides using plasma cleaner
  • Tested chips for blockages in flow
- Three working devices, one with complete blockage in the channel
  • Poured more PDMS and baked overnight at 60°C
  • Followed protocol used in Biomedical Engineering lab class and provided by Dr. Archer

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 AviTagged 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
  1. Add 1μL of Calf Intestine Alkaline Phosphatase (CIAP) to digested vector backbone in order to prevent self-ligation without gene insert included
  2. Incubate at 50°C for 5 minutes
  3. 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 AviTagged 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 AviTagged pZE12 backbone
Ligation
  • GFP + AviTagged Backbone
10.6μL AviTagged pZE12 vector backbone
3.6μL GFP
2.8μL H2O
2.0μL T4 DNA ligase buffer
1.0μL T4 DNA ligase
20μL Total
  • RFP + AviTagged Backbone
10.3μL AviTagged pZE12 vector backbone
6.7μL RFP
2.0μL T4 DNA ligase buffer
1.0μL T4 DNA ligase
20μL Total
  • VioE + AviTagged Backbone
10.6μL AviTagged 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 water bath overnight
Ligation reaction volumes were calculated using a formulated spreadsheet (see Protocol page) 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

Friday, August 5

Lab work done by: Youjin Cho, Claire Paduano

Objective
Transform ligation of GFP/RFP/VioE + AviTagged backbone
  1. Desalted overnight ligation
  2. Transformed the samples into DH5α electrocompetent cells
Group Meeting
  • Just finished transforming GFP/RFP/VioE + AviTag + pZE12 backbone
  • If the VioE gene successfully inserts and ligates with the AviTagged backbone, then we can ligate in VioA and VioB this weekend
  • Goal: to treat PDMS devices to coat with streptavidin and bind AviTagged GFP and RFP to device

Saturday, August 6

Lab work done by: Youjin Cho, Charlie Chung

  • Miniprepped 5mL cultures of transformed DH5α electrocompetent bacteria (standard Qiagen Miniprep protocol)
NOTE: White masses were floating in bacteria culture -- potential contamination?
  • NanoDrop spectrophotometry to determine DNA concentration of 50µL elution product
RFP (Colony 1) + AviTagged pZE12 vector backbone = 53.2ng/µL
RFP (Colony 2) + AviTagged pZE12 vector backbone = 63.4ng/µL
RFP (Colony 3) + AviTagged pZE12 vector backbone = 76.6ng/µL
GFP (Colony 1) + AviTagged pZE12 vector backbone = 65.7ng/µL
GFP (Colony 2) + AviTagged pZE12 vector backbone = 65.5ng/µL
GFP (Colony 3) + AviTagged pZE12 vector backbone = 46.6ng/µL
VioE (Colony 1) did not grow in its culture tube
VioE (Colony 2) + AviTagged pZE12 vector backbone = 53.8ng/µL
DNA from VioE (Colony 3) was accidentally discarded...Charlie is extremely ashamed.