Difference between revisions of "Team:Paris Saclay/Notebook/August/25"

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Agarose gel 1%, migration 90V
 
Agarose gel 1%, migration 90V
 
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[[File:ParisSaclay 25.08.15-Purif sur gel.jpg|300px|center]]
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<html><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_K1707004#5, 3. BBa_K1707022#1, 4. Empty, 5. Empty</i></p></html>
 
We cut corresponding bands with a scalpel.
 
We cut corresponding bands with a scalpel.
  
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* 5 µL H2O
 
* 5 µL H2O
 
* 2 µL Ladder 6x
 
* 2 µL Ladder 6x
 
+
[[File:ParisSaclay 25.08.15-quantif.jpg|300px|center]]
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<html><p><i>Quantification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_R0040 (EcoRI+XbaI), 3. BBa_K1707004#5 (SpeI+EcoRI), 4. BBa_K1707004#5 (SpeI+PstI), 5. BBa_K1707022,  6. BBa_K1707023, 7. Empty, 8. Empty, 9. Empty, 10. Empty</i></p></html>
 
We can conclude:
 
We can conclude:
 
* BBa_K1707022 #1: nothing can be seen
 
* BBa_K1707022 #1: nothing can be seen
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We load on gel 5μL of PCR product.
 
We load on gel 5μL of PCR product.
 
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[[File:ParisSaclay 12.08.15-quantif.jpg|300px|center]]
 +
<html><p><i>Quantification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707020, 3. BBa_K1707030, 4. BBa_I13602#1, 5. BBa_I13602#2,  6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty</i></p></html>
 
We can conclude that we need to start over all PCR except BBa_K115017 which seems to be OK.
 
We can conclude that we need to start over all PCR except BBa_K115017 which seems to be OK.
  

Revision as of 23:09, 18 September 2015


Tuesday 25th August

Lab Work

Electrophoresis purification

by Audrey

  • BBa_K1707004 #5 (digested by SpeI and EcoRI)
  • BBa_K1707022 #1 (digested by XbaI and PstI)

Agarose gel 1%, migration 90V

ParisSaclay 25.08.15-Purif sur gel.jpg

Verification of gel purification, from left to right: 1. DNA Ladder, 2. BBa_K1707004#5, 3. BBa_K1707022#1, 4. Empty, 5. Empty

We cut corresponding bands with a scalpel.

Purification

by Audrey

  • BBa_K1707022 #1
  • BBa_K1707023 #1
  • BBa_K1707004 #5
  • BBa_R0040 #1

With Macherey Nagel purification kit

Quantification

by Audrey

Agarose gel 1%, migration 90V. For each sample:

  • 5 µL plasmid
  • 5 µL H2O
  • 2 µL Ladder 6x
ParisSaclay 25.08.15-quantif.jpg

Quantification, from left to right: 1. DNA Ladder, 2. BBa_R0040 (EcoRI+XbaI), 3. BBa_K1707004#5 (SpeI+EcoRI), 4. BBa_K1707004#5 (SpeI+PstI), 5. BBa_K1707022, 6. BBa_K1707023, 7. Empty, 8. Empty, 9. Empty, 10. Empty

We can conclude:

  • BBa_K1707022 #1: nothing can be seen
  • BBa_K1707023 #1: nothing can be seen
  • BBa_K1707004 #5 (SpeI + EcoRI): 20 µg/µL
  • BBa_K1707004 #5 (SpeI + PstI): 75 µg/µL
  • BBa_R0040 #1: 75 µg/µL

PCR for the Gibson experiment

by Audrey

Amplification Thermometer RNA BBa_K115017

PCR mix for each tube:

  • 36,75 µL H2O
  • 10 µL Buffer Phusion
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer
  • 0,5 μL Reverse primer
  • 1μL plasmid BBa_K115017 v10
  • 0,25 μL DNA Pol Phusion

PCR Cycle:

  • 98°C - 30 seconds
  • 30 cycles:
    • 98°C - 5 seconds
    • 69,8°C - 30 seconds
    • 72°C - 10 seconds
  • 72°C - 10 minutes
  • 4°C for ever

Amplification (reverse PCR)

BioBricks with cI and cI857:

  • BBa_K1707013
  • BBa_K1707019
  • BBa_K1707020
  • BBa_K1707035
  • BBa_K1707036

PCR mix for each tube:

  • 36,75 µL H2O
  • 10 µL Buffer Phusion
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer
  • 0,5 μL Reverse primer
  • 1μL plasmid (dilution 1/10 or 1/100)
  • 0,25 μL DNA Pol Phusion

PCR Cycle:

  • 98°C - 30 seconds
  • 30 cycles:
    • 98°C - 5 seconds
    • 65,6°C - 30 seconds
    • 72°C - 3 minutes
  • 72°C - 10 minutes
  • 4°C for ever

Amplification (reverse PCR)

  • BBa_K1707021

PCR mix for each tube:

  • 36,75 µL H2O
  • 10 µL Buffer Phusion
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer
  • 0,5 μL Reverse primer
  • 1μL plasmid (dilution 1/10 or 1/100)
  • 0,25 μL DNA Pol Phusion

PCR Cycle:

  • 98°C - 30 seconds
  • 30 cycles:
    • 98°C - 5 seconds
    • 65,6°C - 30 seconds
    • 72°C - 3 minutes
  • 72°C - 10 minutes
  • 4°C for ever

Amplification (reverse PCR)

  • BBa_K1707027

PCR mix for each tube:

  • 36,75 µL H2O
  • 10 µL Buffer Phusion
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer
  • 0,5 μL Reverse primer
  • 1μL plasmid (dilution 1/10 or 1/100)
  • 0,25 μL DNA Pol Phusion

PCR Cycle:

  • 98°C - 30 seconds
  • 30 cycles:
    • 98°C - 5 seconds
    • 62,6°C - 30 seconds
    • 72°C - 2 minutes
  • 72°C - 10 minutes
  • 4°C for ever


Electrophoresis

by Audrey

Agarose gel 1%, migration 90V

We load on gel 5μL of PCR product.

ParisSaclay 12.08.15-quantif.jpg

Quantification, from left to right: 1. DNA Ladder, 2. BBa_K1707020, 3. BBa_K1707030, 4. BBa_I13602#1, 5. BBa_I13602#2, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty

We can conclude that we need to start over all PCR except BBa_K115017 which seems to be OK.

Member present:

  • Instructors: Claire
  • Students: Audrey

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