Team:Paris Saclay/Notebook/August/26

Wednesday 26th August

Lab Work

PCR on colony

by Audrey

Biobrick: 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

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

Biobricks:

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

With Macherey-Nagel Extraction kit


Biobricks:

  • 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

Biobricks:

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

With Macherey Nagel purification kit

Reverse PCR

by Audrey

Biobricks:

  • 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


Biobricks:

  • 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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