Team:Paris Saclay/Notebook/August/11

Tuesday 11th August

Lab Work

Ligation

by Pauline

  • BBa_K1707031: BBa_K1707004 + BBa_R0040
    • 10,4 µL BBa_K1707004 digested by EcoRI + SpeI
    • 3,3 µL BBa_R0040 digested by EcoRI + XbaI
    • 2 µL Ligase
    • 2 µL Buffer 10x
    • 2,3 µL H2O
  • BBa_K1707033: BBa_K1707006 + BBa_B0030
    • 7,3 µL BBa_K1707006 digested by XbaII + PstI
    • 7,4 µL BBa_B0030
    • 2 µL Ligase
    • 2,5 µL Buffer 10x
    • 5,8 µL H2O
  • BBa_K1707016: BBa_K1707004 + BBa_I13602
    • 7,2 µL BBa_K1707004 digested by EcoRI + SpeI
    • 2 µL BBa_B0030
    • 2 µL Ligase
    • 1,5 µL Buffer 10x
    • 2,5 µL H2O

Incubation 4°C, 3h

Plamid Extraction

by Pauline

  • BBa_K1707026 #1 and #2
  • BBa_K1707032 #1 and #2

With Macherey-Nagel Extraction kit


Digestion

by Coralie

  • BBa_K1707026 #1 and #2
  • BBa_K1707032 #1 and #2

Mix for each reaction:

  • 0,5 µL PstI
  • 0,5 µL XbaI
  • 1 µL Buffer FastDigest 10x
  • 6 µL H2O
  • 2 µL plasmid

Incubation 37°C, 2h


Electrophoresis

by Pauline

Agarose gel 1%, migration 90V

Biobricks:

  • Quantification:
    • BBa_K1707011 #3 (plasmid)
    • BBa_K1707012 #2 (plasmid)
  • Verification of digestion:
    • BBa_K1707026 #1
    • BBa_K1707026 #2
    • BBa_K1707028 #1
    • BBa_K1707028 #2
    • BBa_K1707032 #1
    • BBa_K1707032 #2
ParisSaclay 11.08.15-vérif+plasmides.jpg

Wells 1-2: Quantification, Wells 4-9: Verification by digestion with XbaI and PstI; from left to right: 1. K1707011#3 plasmid, 2. K1707012#2, 3. DNA Ladder, 4. BBa_K1707026#1, 5. BBa_K1707026#2, 6. BBa_K1707028#1, 7. BBa_K1707028#2, 8. BBa_K1707032#1, 9. K1707032#1, 10. Empty

We can conclude:

  • Quantification:
    • BBa_K1707011 #3 (plasmid): 13 µg/µL
    • BBa_K1707012 #2 (plasmid): 20 µg/µL
  • Verification of digestion:
    • BBa_K1707026 #1: OK but not totally digested
    • BBa_K1707026 #2: OK but not totally digested
    • BBa_K1707028 #1: OK
    • BBa_K1707028 #2: OK
    • BBa_K1707032 #1: OK but not totally digested
    • BBa_K1707032 #2: OK but not totally digested

Digestion

by Coralie

Biobricks:

  • BBa_I13602 x2
    • Mix for each reaction:
      • 1 µL EcoRI
      • 1 µL XbaI
      • 2 µL Buffer FastDigest 10x
      • 6 µL H2O
      • 10 µL plasmid
    • Incubation 37°C, 2h
  • BBa_K1707030 and BBa_K1707020
    • Mix for each reaction:
      • 2 µL Tango Buffer
      • 1 µL SpeI
      • 7 µL H2O
      • 10 µL Plasmid
    • Incubation 2h, 37°C
    • After incubation: we add in each tube:
      • 3 µL Tango Buffer
      • 1 µL EcoRI
      • 1 µL H2O
    • Incubation 1h30, 37°C


Purification on gel

by Pauline

Biobricks:

  • BBa_K1707020
  • BBa_K1707030
ParisSaclay 11.08.15-vérif purif.jpg

Wells 1-2: Quantification, Wells 4-9: Verification by digestion with XbaI and PstI; from left to right: 1. Empty, 2. DNA Ladder, 3. BBa_K1707020, 4. Empty, 5. BBa_K1707030, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty

We can conclude that biobricks are OK

Transformation

by Pauline

Biobricks:

  • BBa_K1707016
  • BBa_K1707031
  • BBa_K1707033

As usual

Low cost experiment

by Coralie

We make 8 plates:

  • In each one:
    • 0,27g Stock Cube
    • 0,600g Agar Agar
    • 0,135g Baker's yeast
    • 45mL mineral water

We make 3 LB plates for control

We use the strain created for the Interlab Study (BBa_J23101 + GFP) to make stries on each plate. We put 3 plates in each condition (2 home-made + 1 LB plate)

  • 37°C
  • In a yogurt maker
  • On the table

Incubation overnight


Member present:

  • Instructors: Claire
  • Students: Coralie and Pauline

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t@iGEMParisSaclay
aUniversité Paris Sud
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