Team:Paris Saclay/Notebook/August/4

Tuesday 4th August

Lab Work

Plasmid extraction

by Pauline

Biobricks:

• BBa_K1707008 #1 to #6
• BBa_I13600 #1 and #2

With the Macherey-Nagel Extraction kit

Digestion Verification

by Pauline and Coralie

Biobricks: BBa_K1707008 #1 to #6

Mix:

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

Incubation 2h, 37°C

Electrophoresis

by Pauline

Agarose gel 1% Migration 90V

We can conclude that all BBa_K1707008 clones are OK We make a glycerol stock of #1 and #2

Electrophoresis

by Pauline and Coralie

Agarose gel, 1%

Migration 100V

We can conclude that:

• BBa_K1707021: #2, #3, #4 and #6 are OK
• BBa_K1707011: #1 to #6 are OK
• BBa_K1707004: #1 to #6 are OK

We make glycerol stock of them.

Digestion

by Coralie

XbaI + PstI:

• BBa_K1707011
• BBa_K1707004 x2
• BBa_E0022
• BBa_E0422
• BBa_K1707012

SpeI + Pst

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

Soil experiment

by Coralie

Water contamination

Sea Water separate in 5mL in 50mL Falcon Fresh water separate in 5mL in 50mL Falcon Contamination with 1mL Water + LB Water + concentrate cells solution Water + concentrate cells solution 10 fold dilution Water + concentrate cells solution 100 fold dilution

After that, we take 100µL on MCK

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

Digestion

by Audrey and Coralie

Biobricks:

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

Mix:

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

Member present:

• Instructors: Claire
• Students: Coralie, Audrey and Pauline

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