Team:Cornell/Week 9
From 2011.igem.org
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July 31st - August 6th
Sunday
Monday
Tuesday
Wednesday
Thursday, August 4
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