Team:Paris Saclay/Notebook/August/10

Monday 10th August

Lab Work

Plasmid extraction

by Pauline

Biobricks:

• BBa_R0051 #1

With Macherey-Nagel Extraction kit

Digestion verification

by Coralie

Biobricks:

• BBa_K1707004 #2
• BBa_K1707011 #1
• BBa_ROO407#1
• BBa_K1707006 #1
• BBa_I13602 #1
• BBa_K1707012 #1
• BBa_R0051 #1
• BBa_S03518 #1

• Mix for BBa_S03518 and BBa_K1707006:
  • 10 µL plasmid
  • 1 µL XbaI
  • 1 µL PstI
  • 2 µL Buffer FastDigest 10x
  • 6 µL H2O

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

• Mix for BBa_R0040 and BBa_I13602:
  • 10 µL plasmid
  • 1 µL EcoRI
  • 1 µL XbaI
  • 2 µL Buffer FastDigest 10x
  • 6 µL H2O

• Mix for BBa_R0051
  • 10 µL plasmid
  • 1 µL SpeI
  • 2 µL PstI
  • 2 µL Tango Buffer 10x
  • 6 µL H2O

Incubation 1h30, 37°C

• Mix for BBa_K1707004:
  • 2 µL Tango Buffer 10x
  • 1 µL SpeI
  • 2 µL H2O
  • 15 µL plasmid

Incubation 1h30, 37°C After incubation, we add:

• • 3 µL Tango Buffer 10x
  • 1 µL EcoRI
  • 1 µL H2O

Incubation 1h30, 37°C

Purification by Electrophoresis

by Coralie

Agarose gel 1%, 100V

Biobricks:

• BBa_K1707004 #1 and #2 (digested by EcoRI + SpeI)
• BBa_K1707011 #1 (digested by XbaI +PstI)
• BBa_K1707006 #1 (digested by XbaI + PstI)
• BBa_K1707012 #1 (digested by XbaI + PstI)
• BBa_S03518 #1 (digested by XbaI + PstI)

We can conclude that:

• BBa_K1707004 #1 and #2 are OK, we can cut the band
• BBa_K1707006 is OK, we can cut the band
• BBa_K1707011: we can't see anything on the gel
• BBa_K1707012: we can't see anything on the gel
• BBa_S03518: is OK, we can cut the band

Purification

by Pauline

Biobricks:

• BBa_K1707004 #2
• BBa_K1707011 #1
• BBa_K1707006 #1
• BBa_K1707012 #1
• BBa_S03518 #1
• BBa_R0040 #1
• BBa_I13602 #1
• BBa_R0051 #1

With Macherey-Nagel Purification kit

Quantification

by Pauline

Biobricks:

• BBa_K1707004 #2 x2
• BBa_K1707006 #1
• BBa_S03518 #1
• BBa_R0040 #1
• BBa_I13602 #1
• BBa_R0051 #1

Agarose gel 1%, migration at 100V

We can conclude:

• BBa_K1707004 #2: 20µg/µL
• BBa_K1707006 #1: 30µg/µL
• BBa_S03518 #1: 50µg/µL
• BBa_R0040 #1: 30 µg/µL
• BBa_I13602 #1: 50µg/µL
• BBa_R0051 #1: we can't see anything

New Culture

by Pauline

Biobricks:

• BBa_K1707026
• BBa_K1707032

We put two clones of each in 5mL LB + 5µL Chloramphenicol

Member present:

• Instructors: Claire
• Students: Coralie and Pauline

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