Difference between revisions of "Team:Paris Saclay/Notebook/August/6"

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(Thursday 6th August)
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[[File:ParisSaclay 06.08.15-Purification.jpg|400px|center]]
 
[[File:ParisSaclay 06.08.15-Purification.jpg|400px|center]]
 
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<html>
<p><i>Verification of gel purification, from left to right: 1. DNA Ladder, 2. BBa_K1707011, 3. BBa_K1707004, 4. BBa_K1707004, 5. BBa_E0022, 6. BBa_E0422, 7. BBa_K1707012, 8. BBa_I13602, 9. Empty, 10. Empty</i></p></html>
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<p><i>Verification of gel purification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707011, 3. BBa_K1707004, 4. BBa_K1707004, 5. BBa_E0022, 6. BBa_E0422, 7. BBa_K1707012, 8. BBa_I13602, 9. Empty, 10. Empty</i></p></html>
 
We made a mistake: BBa_K1707008, BBa_I13602 and Bba_R0051 shouldn't be purified on agarose gel.  
 
We made a mistake: BBa_K1707008, BBa_I13602 and Bba_R0051 shouldn't be purified on agarose gel.  
 
We can observe a big smear for PIJ773.
 
We can observe a big smear for PIJ773.
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Migration 100V
 
Migration 100V
 
[[File:ParisSaclay 06.08.15-Purif.jpg|400px|center]]
 
[[File:ParisSaclay 06.08.15-Purif.jpg|400px|center]]
<html><p><i>Well 1: Verification by digestion with NotI, Wells 3-9: Verification of gel purification; from left to right: 1. BBa_PIJ773, 2. DNA Ladder, 3. BBa_K1707004#1, 4. BBa_K1707008#1, 5. BBa_K1707011#1, 6. BBa_K1707012#1, 7. BBa_R0051, 8. BBa_I13600, 9. BBa_I13602, 10. Empty</i></p></html>
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<html><p><i>Well 1: Verification by digestion with NotI, Wells 3-9: Verification of gel purification; from left to right: 1. BBa_PIJ773, 2. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 3. BBa_K1707004#1, 4. BBa_K1707008#1, 5. BBa_K1707011#1, 6. BBa_K1707012#1, 7. BBa_R0051, 8. BBa_I13600, 9. BBa_I13602, 10. Empty</i></p></html>
  
 
===Ligation===
 
===Ligation===

Revision as of 21:32, 18 September 2015


Thursday 6th August

Lab Work

Soil experiment

by Coralie

Water

Observation of plates (05/08/2015) Almost all plates are yellow. We choose to stop the experiment.

We will define a new protocol:

  • Count of bacteria before contamination
  • Evaluation of the count of bacteria we should see at Day0
  • Measurement of the pH before each sampling (We think that maybe the water pH change with adding the LB)

Low Budget Challenge

by Coralie

We can't say a lot of things... But we can see some colony on the agar with the baker's yeast in the agar. We try another time with different agar recipes:

  • Agar 1:
    • 0,27g Stock Cube
    • 0,600g Agar Agar
    • 0,135g Baker's yeast
    • 0,225g sugar
    • 45mL mineral water
  • Agar 2:
    • 0,27g Stock Cube
    • 0,600g Agar Agar
    • 0,135g Baker's yeast
    • 45mL mineral water

We make striations with BBa_J23101+BBa_I13504 strain with a curved pin. We do the same on an usual LB plate without antibiotics (Control +)

Incubation over night, 37°C


Purification agarose gel

by Pauline and Audrey

  • BBa_K1707012
  • BBa_K1707004
  • BBa_K1707011
  • BBa_R0051
  • BBa_K1707008
  • BBa_I13602
  • BBa_I13600

And PIJ773 digested by NotI

Agarose gel 1%

Migration 90 V

ParisSaclay 06.08.15-Purification.jpg

Verification of gel purification, from left to right: 1. DNA Ladder, 2. BBa_K1707011, 3. BBa_K1707004, 4. BBa_K1707004, 5. BBa_E0022, 6. BBa_E0422, 7. BBa_K1707012, 8. BBa_I13602, 9. Empty, 10. Empty

We made a mistake: BBa_K1707008, BBa_I13602 and Bba_R0051 shouldn't be purified on agarose gel. We can observe a big smear for PIJ773. We can also observe that BBa_I13602 has three bands. Rest is alright.

Purification

by Pauline

  • BBa_K1707011 #1
  • BBa_K1707004 #1
  • BBa_K1707012 #1
  • BBa_R0051
  • BBa_K1707008 #1
  • BBa_I136000
  • BBa_I13602

With Macherey-Nagel Kit


Quantification

by Pauline and Audrey

  • BBa_K1707011 #1
  • BBa_K1707004 #1
  • BBa_K1707012 #1
  • BBa_R0051
  • BBa_K1707008 #1
  • BBa_I136000
  • BBa_I13602

PIJ773 digested by NotI

Agarose gel, 1%

Migration 100V

ParisSaclay 06.08.15-Purif.jpg

Well 1: Verification by digestion with NotI, Wells 3-9: Verification of gel purification; from left to right: 1. BBa_PIJ773, 2. DNA Ladder, 3. BBa_K1707004#1, 4. BBa_K1707008#1, 5. BBa_K1707011#1, 6. BBa_K1707012#1, 7. BBa_R0051, 8. BBa_I13600, 9. BBa_I13602, 10. Empty

Ligation

by Audrey

BBa_K1707026:

  • BBa_E0422 + BBa_J23101

Mix:

  • 11 µL BBa_E0422
  • 8 µL BBa_J23101
  • 1,5 µL Buffer 10x
  • 2 µL Ligase
  • 2,5 µL H2O

BBa_K1707032:

  • BBa_I13600 + BBa_K1707008

Mix:

  • 2 µL BBa_K1707008
  • 5 µL BBa_I13600
  • 1 µL Buffer 10x
  • 2 µL Ligase

BBa_K1707033:

  • BBa_J23101 + BBa_K1707006

Mix:

  • 6,5 µL BBa_K1707006
  • 1,5 µL BBa_J23101
  • 1,5 µL Tampon 10x
  • 2 µL Ligase
  • 3,5 µL H2O

Incubation ON, 4°C

New culture

by Pauline

  • BBa_K1707020
  • Plamsid test
  • BBa_K1707027
  • BBa_R0040
  • BBa_K1707028

2clones in 5mL LB + antibiotics

Member present:

  • Instructors: Claire
  • Students: Coralie, Audrey and Pauline

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