Team:Cornell/Week 7

From 2011.igem.org

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<font size="5"> <i> July 17th - July 23rd <i></font>
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<font size="5"> <i> July 17th - July 23rd </i></font>
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==Sunday==
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==Sunday, July 17==
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Lab work done by: Alyssa Henning & Bill Jo
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Lab work done by: Alyssa Henning, Bill Jo
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*Successfully miniprepped 1 sample of GFP + avitag (the second sample got messed up)
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:*Miniprepped 1 sample of GFP + AviTag (the second sample got messed up)
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*Ran a gel on the RFP that was PCRed on Saturday. We also cut out the bands.
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:*Ran a gel on the RFP that was amplified via PCR on Saturday
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*Sean will innoculate 6 tubes of GFP-containing bacteria--1 tube for us, and 5 tubes for CURIE.
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:*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)
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==Monday==
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==Monday, July 18==
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Lab work done by: Charlie Chung & Youjin Cho
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Lab work done by: Charlie Chung, Youjin Cho
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*Gel purified 2 RFP PCR samples from Friday.
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:*Gel purified 2 RFP PCR samples from Friday
-
*Miniprepped 2 samples of GFP + avitag.
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:*Miniprep 2 samples of GFP + AviTag
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==Tuesday==  
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==Tuesday, July 19==  
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Lab Work Done By: James Mathew
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Lab work done by: James Mathew
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Submitted GFP+avitag genes for synthesis
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:*Submitted GFP + AviTag for synthesis
 +
:*Submitted pZE12 backbone for subcloning of light sensor
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Submitted pZE-12 backbone for subcloning of light sensor
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==Wednesday, July 20==
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Lab work done by: James Mathew
 +
*'''Set up ligation reaction of pZE-12 backbone with the primer dimer insert'''
 +
::1. <u>Digestion of Backbone</u>
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:::- 26µl Backbone plasmid = 2µg DNA
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:::- 5µl NEBuffer 4 (optimal for SphI-HF and ClaI-HF)
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:::- 1µl SphI & 1µl ClaI
 +
:::- 16.5µl H2O
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::::*left in water bath for one hour
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:::- 1µl CIAP added to digestion reaction
 +
::::*left in water bath for 30 minutes
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::2. <u>Ran Digestion Product through gel for 30 minutes at 120V</u>
 +
::::Lane 1: 2µl 1kb Ladder + 2µl 6x loading dye + 8µl H2O
 +
::::Lane 2: 25µl digestion reaction + 5µl 6x loading dye
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::::Lane 3: 25µl digestion reaction + 5µl 6x loading dye
 +
::3. <u>Gel Purification using Qiagen Kit</u>
 +
::4. <u>NanoDrop of Samples</u>
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::5. <u>Ligation Reaction</u>
 +
:*Ligation done overnight for transformation on 7/21/11
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==Wednesday==
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==Thursday, July 21==
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Lab Work Done By: James Mathew
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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
-
Set up ligation reaction of pZE-12 backbone with the primer dimer insert.
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==Friday, July 22==
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  Digestion of Backbone
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[[File:Cornell11 VioA,B,E PCR 7-22.jpg|thumb|Gel electrophoresis]]
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    - 26 ul Backbone plasmid = 2 ug DNA
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<br>
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    - 5 ul Buffer #4 (optimal for SphI-HF and ClaI-HF)
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Lab work done by: James Mathew, Claire Paduano
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    - 1 ul SphI & 1 ul ClaI
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<br><br>
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    - 16.5 ul H20
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*PCR gel purification of VioA, VioB, and VioE
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  left in water bath for one hour
+
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  - 1 ul CIAP added to digestion reaction
+
-
  left in water bath for 30 minutes
+
-
 
+
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  Ran Digestion Product through gel for 30 minutes at 120 V.
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    Lane 1: 2 ul 1kb Ladder + 2 ul 6X Dye + 8 ul H20
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    Lane 2: 25 ul digestion reaction + 5 ul 6X Dye
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    Lane 3: 25 ul digestion reaction + 5 ul 6X Dye
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-
+
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  Gel Purification using Qiagen Kit
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-
+
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  NanoDrop of Samples
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      Concentration:
+
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      Label:
+
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+
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  Ligation Reaction
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      Label:
+
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+
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  Ligation done overnight for transformation on 7/21/11
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==Thursday==
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==Friday==
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==Saturday==
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__NOTOC__
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==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

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 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

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

Gel electrophoresis


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
Retrieved from "http://2011.igem.org/Team:Cornell/Week_7"