Team:Paris Saclay/Notebook/August/13

Thursday 13th August

Lab Work

Plasmid extraction

by Pauline

Biobricks:

• BBa_R0051 (2nd send)
• BBa_K1707011 #3 (x2)
• BBa_K1707012 #2 (x2)
• BBa_K1707033 #1 and #2
• BBa_K1707031 #1 and #2
• BBa_K1707016 #1 and #2

With Macherey-Nagel Extraction kit (BBa_K1707011 #1 and #2 are assembled in the purification column) (BBa_K1707012 #1 and #2 are assembled in the purification column)

Digestion

by Coralie

Biobricks:

• BBa_K1707011
• BBa_K1707012
• BBa_K1707016 #1 and #2
• BBa_K1707031 #1 and #2
• BBa_K1707033 #1 and #2
• BBa_R0051

Mixes:

• BBa_K1707011 and BBa_K1707012
  • 1 µL XbaI
  • 2 µL PstI
  • 2 µL FastDigest Buffer 10x
  • 1 µL H2O
  • 15 µL plasmid

• BBa_K1707016 #1 and #2 and BBa_K1707031 #1 and #2
  • 1 µL XbaI
  • 2 µL PstI
  • 2 µL FastDigest Buffer 10x
  • 11 µL H2O
  • 5 µL plasmid

• BBa_K1707033 #1 and #2
  • 1 µL XbaI
  • 2 µL PstI
  • 2 µL FastDigest Buffer 10x
  • 6 µL H2O
  • 10 µL plasmid

• BBa_R0051
  • 1 µL SpeI
  • 2 µL PstI
  • 2 µL Tango Buffer 10x
  • 5 µL H2O
  • 10 µL plasmid

Incubation 1h30, 37°C

Quantification

by Pauline

Agarose gel 1%, Migration 90V

We can conclude:

• BBa_I13602 #1: 40 µg/µL
• BBa_I13602 #2: 40 µg/µL
• BBa_K1707020: 10 µg/µL
• BBa_K1707030: 13 µg/µL

Digestion

by Coralie

Biobricks:

• BBa_K1707011 #3 and BBa_K1707012 #2
  • Mix for each reaction:
    • 1 µL PstI
    • 1 µL XbaI
    • 2 µL Buffer FastDigest 10x
    • 6 µL H2O
    • 10 µL plasmid
  • Incubation 37°C, 2h

Purification on gel

by Pauline

Agarose gel 1%, 90V

Biobricks:

• BBa_K1707011 #3
• BBa_K1707012 #2

We can conclude that biobricks are OK

Purification by the Macherey-Nagel Purification kit

Ligation

by Pauline

• BBa_K1707035: BBa_K1707030 + BBa_I13602
  • 11,6 µL BBa_K1707030
  • 1,25 µL BBa_R0040
  • 2 µL Ligase
  • 2 µL Buffer 10x
  • 3,15 µL H2O

• BBa_K1707036: BBa_K1707020 + BBa_I13602
  • 15,5 µL BBa_K1707006
  • 1,25 µL BBa_B0030
  • 2 µL Ligase
  • 2,5 µL Buffer 10x
  • 3,75 µL H2O

Incubation 4°C, ON

New culture

by Pauline

Biobricks:

• From stock:
  • BBa_K1707012 #2 x2
  • BBa_K1707011 #3 x2
  • BBa_R0051
• Two clones from plate of each:
  • BBa_K1707033
  • BBa_K1707031
  • BBa_K1707016

In 5mL LB + 5µL of appropriate antibiotic

Member present:

• Instructors: Claire
• Students: Coralie and Pauline

Back to the calendar