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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

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 LadderBBa_K1707004#2, 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

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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