Team:Cornell/Week 7

From 2011.igem.org

(Difference between revisions)
(Saturday)
(Wednesday)
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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.
-
  Digestion of Backbone
+
::1. Digestion of Backbone
-
    - 26 ul Backbone plasmid = 2 ug DNA
+
:::- 26 ul Backbone plasmid = 2 ug DNA
-
    - 5 ul Buffer #4 (optimal for SphI-HF and ClaI-HF)
+
:::- 5 ul Buffer #4 (optimal for SphI-HF and ClaI-HF)
-
    - 1 ul SphI & 1 ul ClaI
+
:::- 1 ul SphI & 1 ul ClaI
-
    - 16.5 ul H20
+
:::- 16.5 ul H20
-
  left in water bath for one hour
+
::::*left in water bath for one hour
-
  - 1 ul CIAP added to digestion reaction
+
:::- 1 ul CIAP added to digestion reaction
-
  left in water bath for 30 minutes
+
::::*left in water bath for 30 minutes
-
  Ran Digestion Product through gel for 30 minutes at 120 V.
+
::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 1: 2 ul 1kb Ladder + 2 ul 6X Dye + 8 ul H20
-
    Lane 2: 25 ul digestion reaction + 5 ul 6X Dye
+
:::Lane 2: 25 ul digestion reaction + 5 ul 6X Dye
-
    Lane 3: 25 ul digestion reaction + 5 ul 6X Dye
+
:::Lane 3: 25 ul digestion reaction + 5 ul 6X Dye
   
   
-
  Gel Purification using Qiagen Kit
+
::3. Gel Purification using Qiagen Kit
   
   
-
  NanoDrop of Samples
+
::4. NanoDrop of Samples
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      Concentration:
+
:::Concentration:
-
      Label:
+
:::Label:
-
  Ligation Reaction
+
::5. Ligation Reaction
-
      Label:
+
:::Label:
*Ligation done overnight for transformation on 7/21/11
*Ligation done overnight for transformation on 7/21/11

Revision as of 16:19, 28 July 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

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

Lab work done by: Charlie Chung & Youjin Cho

  • Gel purified 2 RFP PCR samples from Friday.
  • Miniprepped 2 samples of GFP + avitag.

Tuesday

Lab Work Done By: James Mathew

  • Submitted GFP+avitag genes for synthesis
  • Submitted pZE-12 backbone for subcloning of light sensor

Wednesday

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
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
3. Gel Purification using Qiagen Kit
4. NanoDrop of Samples
Concentration:
Label:
5. Ligation Reaction
Label:
  • 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

Cornell11 VioA,B,E PCR 7-22.jpg

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