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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
- Successfully miniprepped 1 sample of GFP + avitag (the second sample got messed up)
- Ran a gel on the RFP that was PCRed on Saturday. We also cut out the bands.
- Sean will innoculate 6 tubes of GFP-containing bacteria--1 tube for us, and 5 tubes for CURIE.
Monday, July 18
Lab work done by: Charlie Chung & Youjin Cho
- Gel purified 2 RFP PCR samples from Friday.
- Miniprepped 2 samples of GFP + avitag.
Tuesday, July 19
Lab Work Done By: James Mathew
- Submitted GFP+avitag genes for synthesis
- Submitted pZE-12 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
- - 26 ul Backbone plasmid = 2 ug DNA
- - 5 ul Buffer #4 (optimal for SphI-HF and ClaI-HF)
- - 1 ul SphI & 1 ul ClaI
- - 16.5 ul H20
- left in water bath for one hour
- - 1 ul CIAP added to digestion reaction
- left in water bath for 30 minutes
- 1. Digestion of Backbone
- 2. Ran Digestion Product through gel for 30 minutes at 120 V.
- Lane 1: 2 ul 1kb Ladder + 2 ul 6X Dye + 8 ul H20
- Lane 2: 25 ul digestion reaction + 5 ul 6X Dye
- Lane 3: 25 ul digestion reaction + 5 ul 6X Dye
- 2. Ran Digestion Product through gel for 30 minutes at 120 V.
- 3. Gel Purification using Qiagen Kit
- 4. NanoDrop of Samples
- Concentration:
- Label:
- 4. NanoDrop of Samples
- 5. Ligation Reaction
- Label:
- 5. Ligation Reaction
- Ligation done overnight for transformation on 7/21/11
Thursday
Lab work done by: James Mathew
- miniprepped Vio operon (Cambridge 2009)
Lab work done by: Youjin Cho, Charlie Chung, and Alyssa Henning
- Reconstituted VioA, VioB, and VioE forward and reverse primers
- Set up PCR for VioA, VioB, and VioE
- Aborted transformation of pZE-12 plasmid + avitag primer dimer because reverse primers for avitag were incorrect.
- Redesigned reverse primers for avitag to order on Friday
Friday
Lab work done by: James Mathew & Claire Paduano
- PCR gel purification of VioA, VioB, and VioE
Saturday
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
