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Team:Cornell/Week 7
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==Wednesday, July 20== | ==Wednesday, July 20== | ||
Lab work done by: James Mathew | Lab work done by: James Mathew | ||
| - | *'''Set up ligation reaction of pZE-12 backbone with the primer dimer insert | + | *'''Set up ligation reaction of pZE-12 backbone with the primer dimer insert''' |
::1. <u>Digestion of Backbone</u> | ::1. <u>Digestion of Backbone</u> | ||
| - | :::- | + | :::- 26µl Backbone plasmid = 2µg DNA |
| - | :::- | + | :::- 5µl NEBuffer 4 (optimal for SphI-HF and ClaI-HF) |
| - | :::- | + | :::- 1µl SphI & 1µl ClaI |
| - | :::- 16. | + | :::- 16.5µl H2O |
::::*left in water bath for one hour | ::::*left in water bath for one hour | ||
| - | :::- | + | :::- 1µl CIAP added to digestion reaction |
::::*left in water bath for 30 minutes | ::::*left in water bath for 30 minutes | ||
| - | + | ::2. <u>Ran Digestion Product through gel for 30 minutes at 120V</u> | |
| - | ::2. <u>Ran Digestion Product through gel for 30 minutes at | + | ::::Lane 1: 2µl 1kb Ladder + 2µl 6x loading dye + 8µl H2O |
| - | ::::Lane 1: | + | ::::Lane 2: 25µl digestion reaction + 5µl 6x loading dye |
| - | ::::Lane 2: | + | ::::Lane 3: 25µl digestion reaction + 5µl 6x loading dye |
| - | ::::Lane 3: | + | |
| - | + | ||
::3. <u>Gel Purification using Qiagen Kit</u> | ::3. <u>Gel Purification using Qiagen Kit</u> | ||
| - | |||
::4. <u>NanoDrop of Samples</u> | ::4. <u>NanoDrop of Samples</u> | ||
| - | |||
| - | |||
| - | |||
::5. <u>Ligation Reaction</u> | ::5. <u>Ligation Reaction</u> | ||
| - | : | + | :*Ligation done overnight for transformation on 7/21/11 |
| - | + | ||
| - | *Ligation done overnight for transformation on 7/21/11 | + | |
==Thursday, July 21== | ==Thursday, July 21== | ||
Lab work done by: James Mathew | Lab work done by: James Mathew | ||
| - | * | + | *Miniprepped Vio operon (Cambridge 2009) |
| - | + | Lab work done by: Youjin Cho, Charlie Chung, Alyssa Henning | |
| - | Lab work done by: Youjin Cho, Charlie Chung, | + | |
*Reconstituted VioA, VioB, and VioE forward and reverse primers | *Reconstituted VioA, VioB, and VioE forward and reverse primers | ||
*Set up PCR for VioA, VioB, and VioE | *Set up PCR for VioA, VioB, and VioE | ||
| - | *Aborted transformation of | + | *Aborted transformation of pZE12 plasmid + AviTag primer dimer because reverse primers for AviTag were incorrect |
| - | *Redesigned reverse primers for | + | *Redesigned reverse primers for AviTag to order on Friday |
==Friday, July 22== | ==Friday, July 22== | ||
[[File:Cornell11 VioA,B,E PCR 7-22.jpg|thumb|Gel electrophoresis]] | [[File:Cornell11 VioA,B,E PCR 7-22.jpg|thumb|Gel electrophoresis]] | ||
<br> | <br> | ||
| - | Lab work done by: James Mathew | + | Lab work done by: James Mathew, Claire Paduano |
<br><br> | <br><br> | ||
*PCR gel purification of VioA, VioB, and VioE | *PCR gel purification of VioA, VioB, and VioE | ||
==Saturday, July 23== | ==Saturday, July 23== | ||
| - | Lab work done by: James Mathew | + | Lab work done by: James Mathew, Claire Paduano |
| - | *Digestion of VioA, VioB, VioE, GFP, and RFP in preparation for ligation with corrected backbone and | + | *Digestion of VioA, VioB, VioE, GFP, and RFP in preparation for ligation with corrected backbone and AviTag primer dimer |
| - | + | ||
Latest revision as of 17:23, 18 September 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 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
