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Team:Cornell/Week 7
From 2011.igem.org
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July 17th - July 23rd
Sunday, July 17
Lab work done by: Alyssa Henning, Bill Jo
- Miniprepped 1 sample of GFP + AviTag (the second sample got messed up)
- Ran a gel on the RFP that was amplified via PCR on Saturday
- Gel purification and extraction
- Sean will inoculate 6 tubes of GFP-containing bacteria -- 1 tube for us, 5 tubes for CURIE (Cornell iGEM's outreach program)
Monday, July 18
Lab work done by: Charlie Chung, Youjin Cho
- Gel purified 2 RFP PCR samples from Friday
- Miniprep 2 samples of GFP + AviTag
Tuesday, July 19
Lab work done by: James Mathew
- Submitted GFP + AviTag for synthesis
- Submitted pZE12 backbone for subcloning of light sensor
Wednesday, July 20
Lab work done by: James Mathew
- Set up ligation reaction of pZE-12 backbone with the primer dimer insert
- 1. Digestion of Backbone
- - 26µl Backbone plasmid = 2µg DNA
- - 5µl NEBuffer 4 (optimal for SphI-HF and ClaI-HF)
- - 1µl SphI & 1µl ClaI
- - 16.5µl H2O
- left in water bath for one hour
- - 1µl CIAP added to digestion reaction
- left in water bath for 30 minutes
- 2. Ran Digestion Product through gel for 30 minutes at 120V
- Lane 1: 2µl 1kb Ladder + 2µl 6x loading dye + 8µl H2O
- Lane 2: 25µl digestion reaction + 5µl 6x loading dye
- Lane 3: 25µl digestion reaction + 5µl 6x loading dye
- 3. Gel Purification using Qiagen Kit
- 4. NanoDrop of Samples
- 5. Ligation Reaction
- Ligation done overnight for transformation on 7/21/11
- 1. Digestion of Backbone
Thursday, July 21
Lab work done by: James Mathew
- Miniprepped Vio operon (Cambridge 2009)
Lab work done by: Youjin Cho, Charlie Chung, Alyssa Henning
- Reconstituted VioA, VioB, and VioE forward and reverse primers
- Set up PCR for VioA, VioB, and VioE
- Aborted transformation of pZE12 plasmid + AviTag primer dimer because reverse primers for AviTag were incorrect
- Redesigned reverse primers for AviTag to order on Friday
Friday, July 22
Lab work done by: James Mathew, Claire Paduano
- PCR gel purification of VioA, VioB, and VioE
Saturday, July 23
Lab work done by: James Mathew & Claire Paduano
- Digestion of VioA, VioB, VioE, GFP, and RFP in preparation for ligation with corrected backbone and avitag primer dimer
