Wednesday 22nd July

Lab Work

Soil experiment

by Audrey, Seong-Koo and Johan

Plates observation and count of CFU:

• Strain 1696:
  • Dilution 1: 490 CFU
  • Dilution 0.1: 67 CFU
  • Dilution 0.01: 0 CFU

• Strain 1693:
  • Dilution 1: 1308 CFU
  • Dilution 0.1: 196 CFU
  • Dilution 0.01: 0 CFU

• Strain 1320:
  • Dilution 1: 186 CFU
  • Dilution 0.1: 35 CFU
  • Dilution 0.01: 1 CFU

We take 1g of contaminated soil and treat it like yesterday to spread it on specific plates Incubation ON, 37°C

Digestion

by Coralie

Biobrick: BBa_K1707003 #4 to #9

• Digestion by EcoRI: mix
  • 6µL EcoRI
  • 6µL Buffer FastDigest 10x
  • 36µL H2O

We put 8µL of the mix in each tube + 2µL of our plasmids

• Digestion by EcoRI + PstI: mix
  • 6µL EcoRI
  • 6µL PstI
  • 6µL Buffer FastDigest 10x
  • 30µL H2O

We put 8µL of the mix in each tube + 2µL of our plasmids

Incubation 1h30, 37°C

Electrophoresis

by Johan

Agarose gel: 1% Migration 80V Biobricks: BBa_K1707003 #4 to #9 digested by EcoRI or EcoRI+PstI

We can't conclude anything because we changed #4 in each gel and we forgot the control

Digestion

by Coralie

Biobricks: BBa_K1707003 #4 and #5 and BBa_B0015

• Digestion by EcoRI: mix
  • 3µL EcoRI
  • 3µL Buffer FastDigest 10x
  • 18µL H2O

We put 8µL of the mix in each tube + 2µL of our plasmids

• Digestion by EcoRI + PstI: mix
  • 3µL EcoRI
  • 3µL PstI
  • 3µL Buffer FastDigest 10x
  • 15µL H2O

We put 8µL of the mix in each tube + 2µL of our plasmids

Incubation 1h30, 37°C

Electrophoresis

by Coralie

Agarose gel: 1% Migration 80V Biobricks: BBa_K1707003 #4 and #5 and BBa_B0015 digested by EcoRI or EcoRI+PstI

We can confirm the BBa_K1707003 #4 and #5

Gel purification

by Audrey

Biobricks:

• BBa_K1707002
• BBa_K098997
• BBa_C0051

Agarose gel 1%, migration 70V

We cut bands with a scalpel

Purification

by Audrey

Biobricks:

• BBa_K098997
• BBa_B0015
• BBa_C0051
• BBa_K1707002
• BBa_K1707001

With PCR Clean up/Gel extraction kit from Macherey Nigel

Quantification and Verification

by Audrey

Biobricks:

• Quantification:
  • BBa_K098997
  • BBa_B0015
  • BBa_C0051
  • BBa_K1707002
  • BBa_K1707001
• Verification:
  • BBa_K1707000

Agarose gel 1% Migration 110V

We can conclude that the BBa_K1707000 PCR is ok. So the biobrick is confirmed

Ligation

by Coralie

• BBa_K1707006
  • 3µL BBa_C0051
  • 2µL BBa_B0015
  • 1µL Ligase
  • 1µL Buffer 10x
  • 3µL H2O

• BBa_K1707007
  • 8µL BBa_K098997
  • 2µL BBa_B0015
  • 1µL Ligase
  • 2µL Buffer 10x
  • 7µL H2O

• BBa_K1707005
  • 13µL BBa_K1707002
  • 2µL BBa_K1707001
  • 1µL Ligase
  • 2µL Buffer 10x
  • 2µL H2O

Incubation ON, 4°C

Member present:

• Instructors:Alice
• Students: Audrey, Coralie, Pauline, Seong-Koo, Johan

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