Team:Cornell/Week 7

From 2011.igem.org

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==Wednesday, July 20==
==Wednesday, July 20==
Lab work done by: James Mathew
Lab work done by: James Mathew
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*'''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>
-
:::- 26 ul Backbone plasmid = 2 ug DNA
+
:::- 26µl Backbone plasmid = 2µg DNA
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:::- 5 ul Buffer #4 (optimal for SphI-HF and ClaI-HF)
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:::- 5µl NEBuffer 4 (optimal for SphI-HF and ClaI-HF)
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:::- 1 ul SphI & 1 ul ClaI
+
:::- 1µl SphI & 1µl ClaI
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:::- 16.5 ul H20
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:::- 16.5µl H2O
::::*left in water bath for one hour
::::*left in water bath for one hour
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:::- 1 ul CIAP added to digestion reaction
+
:::- 1µl CIAP added to digestion reaction
::::*left in water bath for 30 minutes
::::*left in water bath for 30 minutes
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+
::2. <u>Ran Digestion Product through gel for 30 minutes at 120V</u>
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::2. <u>Ran Digestion Product through gel for 30 minutes at 120 V.</u>
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::::Lane 1: 2µl 1kb Ladder + 2µl 6x loading dye + 8µl H2O
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::::Lane 1: 2 ul 1kb Ladder + 2 ul 6X Dye + 8 ul H20
+
::::Lane 2: 25µl digestion reaction + 5µl 6x loading dye
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::::Lane 2: 25 ul digestion reaction + 5 ul 6X Dye
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::::Lane 3: 25µl digestion reaction + 5µl 6x loading dye
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::::Lane 3: 25 ul digestion reaction + 5 ul 6X Dye
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-
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::3. <u>Gel Purification using Qiagen Kit</u>
::3. <u>Gel Purification using Qiagen Kit</u>
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::4. <u>NanoDrop of Samples</u>
::4. <u>NanoDrop of Samples</u>
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::::Concentration:
 
-
::::Label:
 
-
 
::5. <u>Ligation Reaction</u>
::5. <u>Ligation Reaction</u>
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::::Label:
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:*Ligation done overnight for transformation on 7/21/11
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+
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*Ligation done overnight for transformation on 7/21/11
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==Thursday, July 21==
==Thursday, July 21==
Lab work done by: James Mathew  
Lab work done by: James Mathew  
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*miniprepped Vio operon (Cambridge 2009)
+
*Miniprepped Vio operon (Cambridge 2009)
-
 
+
Lab work done by: Youjin Cho, Charlie Chung, Alyssa Henning  
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Lab work done by: Youjin Cho, Charlie Chung, and Alyssa Henning  
+
*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 pZE-12 plasmid + avitag primer dimer because reverse primers for avitag were incorrect.
+
*Aborted transformation of pZE12 plasmid + AviTag primer dimer because reverse primers for AviTag were incorrect
-
*Redesigned reverse primers for avitag to order on Friday
+
*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>
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Lab work done by: James Mathew & Claire Paduano
+
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 & Claire Paduano
+
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  
+
*Digestion of VioA, VioB, VioE, GFP, and RFP in preparation for ligation with corrected backbone and AviTag primer dimer
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__NOTOC__
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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