Difference between revisions of "Team:Paris Saclay/Notebook/August/26"

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==Lab Work==
 
==Lab Work==
  
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===Electrophoresis===
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''by Audrey''
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Agarose gel 1%, migration 90V
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We load on gel 5μL of PCR product.
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[[File:ParisSaclay_26.08.15-_PCR2.jpg|300px|center]]
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<html><p><i>Verification of PCR products, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707035 (10%), 3. BBa_K1707035 (1%), 4. BBa_K1707036 (10%), 5. BBa_K1707036 (1%),  6. BBa_K1707021 (10%), 7. BBa_K1707021 (1%), 8. BBa_K1707027 (10%), 9. BBa_K1707027 (1%), 10. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 11. Empty, 12. Empty</i></p></html>
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[[File:ParisSaclay 26.08.15- PCR1.jpg|300px|center]]
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<html><p><i>Verification of PCR products, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707013 (10%), 3. BBa_K1707013 (1%), 4. BBa_K1707019 (10%), 5. BBa_K1707019 (1%),  6. BBa_K1707020 (10%), 7. BBa_K1707020 (1%), 8. BBa_K1707030 (10%), 9. BBa_K1707030 (1%), 10. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a></i></p></html>
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[[File:ParisSaclay 26.08.15- PCR3.jpg|300px|center]]
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<html><p><i>Verification of PCR products, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K115017, 3. BBa_K115017, 4. BBa_K115017, 5. BBa_K115017</a></i></p></html>
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 +
We can conclude that we need to start over all PCR except BBa_K115017 which seems to be OK.
 
===PCR on colony===
 
===PCR on colony===
 
''by Audrey''
 
''by Audrey''
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PCR Products of BBa_K1707037
 
PCR Products of BBa_K1707037
 +
 +
[[File:ParisSaclay 26.08.15- PCR colo.jpg|300px|center]]
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<html><p><i>Verification of K1707037 colony PCR, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. #1, 3. #2, 4. #3, 5. #4,  6. #5, 7. #6, 8. #7, 9. #8, 10. #9</i></p></html>
  
 
We can't conclude anything because we can't see any positive result. So we choose arbitrary 5 clones for different extractions.
 
We can't conclude anything because we can't see any positive result. So we choose arbitrary 5 clones for different extractions.
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'' by Audrey''
 
'' by Audrey''
  
#1
+
First
 
* BBa_K1707013
 
* BBa_K1707013
 
* BBa_K1707019
 
* BBa_K1707019
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* With and without DMSO 1,5μL
 
* With and without DMSO 1,5μL
  
#2
+
Second
 
* BBa_K1707021
 
* BBa_K1707021
 
* BBa_K1707027
 
* BBa_K1707027

Latest revision as of 23:31, 18 September 2015


Wednesday 26th August

Lab Work

Electrophoresis

by Audrey

Agarose gel 1%, migration 90V

We load on gel 5μL of PCR product.

ParisSaclay 26.08.15- PCR2.jpg

Verification of PCR products, from left to right: 1. DNA Ladder, 2. BBa_K1707035 (10%), 3. BBa_K1707035 (1%), 4. BBa_K1707036 (10%), 5. BBa_K1707036 (1%), 6. BBa_K1707021 (10%), 7. BBa_K1707021 (1%), 8. BBa_K1707027 (10%), 9. BBa_K1707027 (1%), 10. DNA Ladder, 11. Empty, 12. Empty

ParisSaclay 26.08.15- PCR1.jpg

Verification of PCR products, from left to right: 1. DNA Ladder, 2. BBa_K1707013 (10%), 3. BBa_K1707013 (1%), 4. BBa_K1707019 (10%), 5. BBa_K1707019 (1%), 6. BBa_K1707020 (10%), 7. BBa_K1707020 (1%), 8. BBa_K1707030 (10%), 9. BBa_K1707030 (1%), 10. DNA Ladder

ParisSaclay 26.08.15- PCR3.jpg

Verification of PCR products, from left to right: 1. DNA Ladder, 2. BBa_K115017, 3. BBa_K115017, 4. BBa_K115017, 5. BBa_K115017

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

PCR on colony

by Audrey

  • BBa_K1707037 #1 to #9

PCR mix for each tube:

  • 9,75 µL H2O
  • 5 µL Buffer Taq 10x
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer iPS43
  • 0,5 μL Reverse primer iPS44
  • 3 μL 25mM MgCl2
  • 0,25 μL Taq Pol

PCR Cycle:

  • 95°C - 6 + 2 min
  • 30 cycles:
    • 95°C - 30 seconds
    • 51,8°C - 30 seconds
    • 72°C - 2 minutes
  • 72°C - 10 minutes
  • 4°C for ever


Electrophoresis

by Audrey

Agarose gel 1%, migration 90V

PCR Products of BBa_K1707037

ParisSaclay 26.08.15- PCR colo.jpg

Verification of K1707037 colony PCR, from left to right: 1. DNA Ladder, 2. #1, 3. #2, 4. #3, 5. #4, 6. #5, 7. #6, 8. #7, 9. #8, 10. #9

We can't conclude anything because we can't see any positive result. So we choose arbitrary 5 clones for different extractions.


Electrophoresis purification

by Audrey

PCR product of BBa_K175017 from the 08/25/2015

In each weel of the agarose gel, we put 45μL of PCR Product + 15 μL Ladder 6x

Agarose gel 1%, Migration 90V

We cut bands with a scalpel and purificate them by the Macherey-Nagel gel purification kit


Plasmid extraction

by Audrey

  • BBa_K1707022 #1
  • BBa_K1707023 #2
  • BBa_K1707037 #5 to #9
  • BBa_K1707004 #2 (from two different cultures)

With Macherey-Nagel Extraction kit

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

Agarose gel 1%, migration 90V

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

Reverse PCR

by Audrey

First

  • BBa_K1707013
  • BBa_K1707019
  • BBa_K1707020
  • BBa_K1707030
  • BBa_K1707035
  • BBa_K1707036

PCR mix for each tube:

  • 36,75 µL H2O
  • 10 µL Buffer Phusion 5x
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer insertion cI
  • 0,5 μL Reverse primer RV
  • 1μL plasmid
  • 0,25 μL DNA Pol Phusion
  • With and without DMSO 1,5μL

Second

  • BBa_K1707021
  • BBa_K1707027

PCR mix for each tube:

  • 36,75 µL H2O
  • 10 µL Buffer Phusion 5x
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer insertion CFP or GFP
  • 0,5 μL Reverse primer insertion RV
  • 1μL plasmid
  • 0,25 μL DNA Pol Phusion
  • With and without DMSO 1,5μL


Control (-) for primers:

PCR mix for each tube:

  • 36,75 µL H2O
  • 10 µL Buffer Phusion 5x
  • 1 μL dNTP 10mM
  • 0,5 μL Forward primer insertion CFP/GFP/cI + 0,5μL iPS44 or 0,5 μL Reverse primer insertion RV + 0,5μL iPS43
  • 1μL plasmid BBa_K1707013 or BBa_K1707021 or BBa_K1707027
  • 0,25 μL DNA Pol Phusion
  • With and without DMSO 1,5μL


PCR Cycle:

  • 98°C - 30 seconds
  • 3 cycles:
    • 98°C - 5 seconds
    • 55°C - 30 seconds
    • 72°C - 3 minutes
  • 27 cycles:
    • 98°C - 5 seconds
    • 65°C - 30 seconds
    • 72°C - 3 minutes
  • 72°C - 10 minutes
  • 4°C for ever

Member present:

  • Instructors: Claire
  • Students: Audrey

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