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)
ParisSaclay 10.08.15-vérif purif.jpg

Verification of gel purification, from left to right: 1. Empty, 2. DNA Ladder, 3. BBa_K1707004#1, 4. BBa_K1707004#2, 5. BBa_K1707006, 6. BBa_K1707011, 7. BBa_K1707012, 8. BBa_S03518, 9. Empty, 10. Empty

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

Paris Saclay-10.08.2015 - Quantification 2.jpg

Quantification, from left to right: 1. DNA Ladder, 2. BBa_K1707004#1, 3. BBa_K1707004#2, 4. BBa_K1707006, 5. BBa_I13602, 6. BBa_S03518, 7. BBa_R0040, 8. BBa_R0051, 9. Empty, 10. Empty, 11. Empty, 12. Empty

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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