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

Water

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

Incubation ON 37°C

Measurment

'By Johan'

Tecan utilisation :

This time, we use LB and chloramphenicol because the plate has just a protection and we suspect a contamination of our samples.

We depose in inch well 300µL

We analyse the OD and fluorescence's variation (the excitation and emission wave lenght were choose after a scan in the process to obtain the best results)

For each sample, we depose twelve time (12x8 plate)

• LB
• Competent cells
• Cells with J23101
• Cells with J23101 + GFP
• Cells with J23106
• Cells with J23106 + GFP
• Cells with J23117
• Cells with J23117 + GFP

We let's run for 20 cycles of 1 hour

Flow cytometric analysis We analyse the sample see previously with ODmax and OD 0.4 But we doesn't use the good scale so we will reused it tomorrow

Member present:

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

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