Team:Paris Saclay/Notebook/August/1

Monday 3rd August

Lab Work

New culture

by Pauline

Biobricks:

• BBa_K1707008
• BBa_I13600 (2014)

6 clones of each in 5mL LB + 20µg/mL of chloramphenicol

Incubation at 37°C, 250 rpm

Plasmid extraction

by Seong-Koo

Biobricks:

• BBa_K1707011 #1 to #6
• BBa_K1707021 #1 to #6
• BBa_K1707004 #1 to #6

Plasmid pIJ773

With the Macherey-Nagel Extraction kit

Soil experiment

by Coralie

Soil

Observation of plates (31/07/2015):

• Soil + H2O on new MCK: lots of colonies are visible
• Soil + LB on new MCK: lots of colonies are visible
• LB on new MCK: nothing
• MCK alone: nothing

We can suppose two different hypothesis:

• The soil is contaminated
• the MCK isn't OK for our experiment: now we add lactose in the preparation. We will try with sorbitol instead of lactose.

We try soil + H2O and soil +LB on:

• MCK + Sorbitol: Without antibiotics, with Spectinomycine, Tetramycine or Chloramphenicol
• MCK + Lactose: Without antibiotics, with Spectinomycine, Tetramycine or Chloramphenicol

Incubation ON, 37°C

Rehydratation

by Pauline

Biobrick: BBa_I13600 (2014-3-19C and 2013-3-20C)

Transformation

by Pauline

Biobricks:

• BBa_I13600 (2013, 2014 and 2015)
• BBa_K1707008

As usual

Plasmid extraction

by Seong-Koo

Biobricks:

• BBa_E0422 #1 and #2
• BBa_E0022 #1 and #2

With the Macherey-Nagel Extraction kit

New culture

by Pauline

Biobricks:

• BBa_K1707004: amicillin
• BBa_K1707011
• BBa_K1707021

6 clones of each in 5 mL LB

Digestion Verification

by Coralie

• Biobricks:
  • BBa_K1707012 #1, #2, #3 and #4
  • BBa_K1707013 #1, #2, #3 and #4
  • BBa_K1707019 #1, #2, #3 and #4

• Mix:
  • 1 µL Buffer FastDigest 10x
  • 0,5 µL XbaI
  • 0,5 µL PstI
  • 2 µL Plasmid
  • 6 µL H2O

Incubation 2h, 37°C

Electrophoresis

by Coralie

Agarose gel, 1% Migration 100V

We can conclude that all biobricks are OK

Soil experiment

by Coralie and Johan

Soil

Observation of J0 plates: there are a lot of colony in each.

Control (-):

• MCK + Tet: 40 pink colonies
• MCK + Cm: 10 pink colonies
• MCK + Spectino: 1 pink colony
• MCK: 100 pink colonies and a lot of white colonies

We can suppose that we have a contamination between our experiment and control. Or maybe we have a MCK problem because we change it during the experiment.

So, we decide to stop the experiment and do some test:

• Soil + H2O on new MCK
• Soil + LB on new MCK
• LB on new MCK
• MCK alone

Incubation over night, 37°C

New culture

by Pauline

Biobricks:

• BBa_K1707005
• BBa_I13602

A plasmid: PIJ773

Incubation at 37°C, 250 rpm

Member present:

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

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