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

Biobricks:

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

With Macherey-Nagel Extraction kit

Digestion

by Coralie

Biobricks:

• 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

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 of home

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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