Tuesday 21st July

Lab Work

PCR

Pauline PCR Mix:

• Buffer (5x): 30µL
• Forward Primer: 7,5 µL
• Reverse Primer: 7,5µL
• dNTP: 3µL
• DNApol GoTaq: 0,75µL
• MgCl2: 15µL
• H2O: 63,75µL

2µL of DNA in each tube:

• BBa_J23101
• BBa_K1707000

18µL from the mix in each tube

Cycle:

• Initiation: 95°C, 2min
• Cycle: 95°C, 1min - 61°C, 1min - 72°C, 20sec
• Termination: 72°C, 5min

New culture

by Pauline

• BBa_C0051
• BBa_E0240
• BBa_J06702

200µL in 5mL LB + Antibiotic

• 1696 - Bad::Tetracyclin
• 1320 - ClpP::Spectinomycine
• 1693 - MG5516Z1

Plasmid extraction

by Coralie and Audrey

• BBa_K1707001 #1 and #2
• BBa_K1707002 #1 and #2
• BBa_K1707003 #1 and #2
• BBa_R0051
• BBa_K098997

Digestion

by Coralie and Johan In each tube:

• 10 µL plasmid
• 1µL of each enzyme
• 1 µL FastDigest Buffer (10x)
• 6 µL H2O

• BBa_K1707001: SpeI and EcoRI
• BBa_K1707002: XbaI and PstI
• BBa_K1707003 #1 and #2: XbaI and PstI
• BBa_J23101: SpeI and PstI
• BBa_R0051: SpeI and PstI
• BBa_K098997: EcoRI and SpeI

Incubation 1h30, 37°C

Observation plates from soil experiment

by Seong-Koo and Johan

On all MacConkey plates: we can't see anything On LB+antibiotics plates: we can't see anything On LB plates without antibiotics with 10^-5 and 10^-6 dilutions: we can't see anything On LB plates without antibiotics with 10^-2, 10^^-3 and 10^-4 dilutions: we see some colonies

Purification on electrophoresis gel

by Johan and Audrey

• BBa_K1707002
• BBa_K1707003 #1 and #2
• BBa_K098997

Agarose gel, 1% Migration: 80V

BBa_K1707003: the digestion doesn't work. We will try other clones from the transformation

We cut bands of BBa_K1707002 and BBa_K098997 with a scalpel.

DNA Purification

by Audrey

• BBa_K1707002
• BBa_K098997
• BBa_J23101
• BBa_R0051
• BBa_K1707001

With PCR Clean-Up / Gel extraction from Macherey Nigel

New Culture

by Coralie

• BBa_K1707003

We put in culture 10 clones of this Biobrick in 2mL LB + 2µL Cm

Soil Experiment

by Johan and Coralie We put 5g of soil samples in plates.

We mesure OD600 of our cultures:

• 1320 - ClpP::Cm : OD600=1,32
• 1696 - Bad::tetra : OD600=1,12
• 1693 MG1655Z1 : OD600=1.25

In each plate, we put 1mL of one of this strain:

• 1320
• 1696
• 1693

in one of this dilution: 1, 0,1 or 0,01

At t=0, we take 1g of contamined soil, we mixed it with 5mL of steril water, we take the supernatant, dilute it at 10^-2, and put 100µL on each MacConkey plate with the right antibiotic to allow each strain to growth.

We incubate plates at 37°C one night

Control +: We put 100µL of each strain directly on their right plate, without touching the soil Control -: We extract the soil without any contamination and put 100µL of the 10^-2 dilution on a MacConkey plate

Member present:

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

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