Team:Paris Saclay/Notebook/July/29

Wednesday 29th July

Lab Work

Plasmid extraction

by Seong-Koo

Biobricks:

• BBa_K1707008 #1 and #2
• BBa_K1707010 #1 and #2
• BBa_R0051

With the Macherey-Nagel Extraction kit

Digestion

by Pauline

• BBa_K1707008 #1
  • 2µL Buffer FastDigest 10x
  • 1µL SpeI
  • 1µL PstI
  • 10µL Plasmid
  • 6µL H2O

• BBa_K1707010 #1
  • 2µL Buffer FastDigest 10x
  • 1µL XbaI
  • 1µL PstI
  • 10µL Plasmid
  • 6µL H2O

• BBa_R0051
  • 1µL Buffer FastDigest 10x
  • 1µL SpeI
  • 1µL PstI
  • 2µL Plasmid
  • 5µL H2O

Incubation 1h30, 37°C

Purification gel

by Pauline

Biobricks:

• BBa_K1707010 #1

Purification on agarose gel 1%, migration 100V Cut with a scalpel

Purification

by Pauline

Biobricks:

• BBa_K1707008 #1
• BBa_K1707010 #1
• BBa_R0051

Purification with the PCR Clean up kit from Macherey-Nagel

Digestion for verification

by Audrey

Biobricks:

• BBa_K1707005 #1 and #2

Mix:

• 2µL plasmid
• 0,5µL NotI
• 1µL Buffer FastDigest 10x
• 6,5µL H2O

Incubation 1h30, 37°C

Quantification

by Pauline

Biobricks:

• BBa_K1707003 #5
• BBa_K1707008 #1
• BBa_K1707010 #1
• BBa_R0051

Agarose gel, 1%, migration 110V

We can conclude that K1707005 isn't digested

We can conclude the quantification:

• BBa_K1707003 #5: 10ng/µL
• BBa_K1707008 #1: not OK
• BBa_K1707010 #1: 10 ng/µL
• BBa_R0051: 8ng/µL

Ligation

by Audrey

• BBa_K1707012: BBa_B0030 + BBa_K1707007
  • 6 µL BBa_B0030
  • 13 µL BBa_K1707007
  • 2,5µL Buffer Ligase 10x
  • 1 µL Ligase
  • 2,5 µL H2O

• BBa_K1707019: BBa_K1707000 + BBa_K1707006
  • 2,5µL BBa_K1707000
  • 13 µL BBa_K1707006
  • 2 µL Buffer Ligase 10x
  • 1 µL Ligase
  • 1,5 µL H2O

• BBa_K1707013: BBa_K1707000 + BBa_K1707007
  • 1 µL BBa_K1707000
  • 6 µL BBa_K1707007
  • 1 µL Buffer Ligase 10x
  • 1 µL Ligase
  • 1 µL H2O

• BBa_K1707009: BBa_S03518 + BBa_R0051
  • 5,5µL BBa_S03518
  • 6,5 µL BBa_R0051
  • 1,5µL Buffer Ligase 10x
  • 1 µL Ligase
  • 0,5 µL H2O

• BBa_K1707004: BBa_K1707003 + BBa_R0051
  • 10 µL BBa_K1707003
  • 6,5 µL BBa_K1707007
  • 2 µL Buffer Ligase 10x
  • 1 µL Ligase
  • 0,5 µL H2O

Incubation over night, 16°C

Transformation

by Coralie

Biobricks:

• BBa_K1707012
• BBa_K1707019
• BBa_K1707013

As usual

Rehydratation and transformation

by Johan

Biobricks:

• BBa_E0022
• BBa_E0422

New Culture

by Pauline

Biobricks:

• BBa_K1707008 #3, #4, #5 and #6

Soil experiment

by Johan and Coralie

Soil

Observation of J0 plates: there are a lot of colony in each. Contamination is alright. Controls (+) and (-) are Ok too. We take 1g of each pot of soil and we dilute it in 5mL sterile H2O. After shaking and decant, we put 100µL of supernatant on the right plate. Incubation ON 37°C

We can't see anything on water plates. We try another type, like yesterday but without dilution and dilution 10-1. Incubation ON 37°C

_

Soil experiment

by Audrey, Johan and Coralie

Soil

We put 100µL of each culture on MacConkey plate with specific antbiotic (Control +):

• 1696: Tetracyclin
• 1693: Spectinomycine
• 1320: Chloramphenicol

We put 30 mL of each culture in 60g of non sterile soil in a pot.

• 1696: 3 pots
• 1693: 3 pots
• 1320: 3 pots

We put 30 mL of clean LB in 60g of non sterile soil in a pot (Control -)

• LB: 3 pots

After this contamination, we take 1g of each pot to make the J0 measurement. We dilute it in 5mL of sterile H2O, and shake it during 5 min. Then, we let it to decant during 15 min. We take 50µL of the supernatant, and dilute it in 950µL of Sterile H2O (complete dilution from the soil is now at 10-2) We put 100µL of this previous dilution on plates that fits. We incubate plates over night at 37°C

Pots are placed in trays: 1 strain by tray. Trays are covered by a shrink-wrap, and we let them in room temperature

Water

We test two different water: seawater, from the Atlantic ocean, and stagnant water from Orsay's pool. For each water, we make dilutions: 10-1, 10-2, 10-3 and 10-4 We 100µL of different dilution on each type of plate:

• LB without antibiotic
• LB + Spectinomycin
• LB + tetracyclin
• LB + Chloramphenicol
• MacConkey without antibiotic
• MacConkey + Spectinomycin
• MacConkey + Tetracyclin
• MacConkey + Chloramphenicol

We incubate plates over night at 37°C

New culture

by Pauline

Biobricks:

• BBa_K1707008
• BBa_K1707010

We take 2 colony of each, and put it in 5mL of LB + 20µg/mL Chloramphenicol Incubation 37°C, 250 rpm

Purification

by Audrey

Biobricks:

• BBa_B0030
• BBa_R0051
• BBa_J23117 #1 and #2
• BBa_K1399019
• BBa_K1399023
• BBa_K1707006
• BBa_K1707007

With PCR CLean-up/Gel extraction kit from Macherey-Nigel

Quantification

by Audrey

Agarose gel 1% Migration 100V

Interlab study

by Johan

We try to use the TEKAN to mesure our fluorescence, but it doesn't work.

We create new culture of BBa_J23101/BBa_J23106/BBa_J23117 + BBa_I13504.

Member present:

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

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