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From 2014.igem.org

Contents 1 Tuesday 22st July 1.1 Lab Work 1.1.1 1 - Extraction of p cola plasmid DNA 1.1.2 2 - Samples for stock 1.1.3 3 - Electrophoresis 1.1.4 4 - Bacterial Culture 1.1.5 5 - Plasmid DNA extraction 1.2 C - Lemon scent 1.2.1 PCR Targeting

Tuesday 22st July

Lab Work

1 - Extraction of p cola plasmid DNA

by Sean

p cola is a low-copy plasmid, and thus requires a slightly different protocol than other plasmids.

2 - Samples for stock

by Fabio

We collected 1ml of each sample, added 240µl of glycerol (87%) and stored at -80°C.

• BBa_K1033902
• BBa_K1033905
• BBa_K1033910
• BBa_K1033913
• BBa_K1033922
• BBa_K1033925
• BBa_K1033927
• BBa_K1033929
• BBa_K103921
• BBa_J45017
• BBa_K731201
• BBa_K762100

from Liquide Bacterial Cultures transformed the 21st July

3 - Electrophoresis

by Arnaud (Process B), Fabio (gel), Marie and Romain (Process B)

We used 2µL of DNA of the following Biobricks in a 1% Agarose Gel.

Process A

Process B

Results:

• A:
• B:
• C:

Protocol

4 - Bacterial Culture

by Fabio and Marie

One liquid culture with 5ml of LB (37°C - 150 rpm), IL1403 from the concentrated colony.

5 - Plasmid DNA extraction

by Sean and Terry

Plasmids used: cf part 1

Note: we accidentally used a buffer without 10% ethanol. After removal of the buffer, we repeated the step with a correct version. This may have affected the electrophoresis which followed.

C - Lemon scent

PCR Targeting

by Romain

Strains used: DY330 (clone I and II). Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli)

Protocol: Step 1 - Dilution of 300µl of bacterial culture in 30ml of LB + 30µl Cm at 30°C.

Step 2 - With the pre-culture:

• make glycerol stock: 1ml bacterial culture with 240µl glycerol 87%.
• Make extraction of 5ml of plasmid Protocol

Step 3 - When the culture OD₆₅₀ = 0,6 (Step 1):

• put in ice during 10min.
• centriguge 4°C, 5min, 4000rpm.
• Discard the supernatant, and resuspend the pellet in 30ml glycerol 10% COLD.
• centriguge 4°C, 5min, 4000rpm.
• Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% COLD.
• centriguge 4°C, 5min, 4000rpm.
• Discard the supernatant, and resuspend the pellet in 200µl glycerol 10% COLD.

Step 4 - Transformation:

• control 50µl (without DNA)
• 50µl transformed bacteria + 2,5µl plasmid + 7,5µl pOSV 230 PCR (purifié)

Step 5 - Electroporation control

Step 6 - Spread on ApraR and CmR dishes

• Control dishes : 100µl ND
• 250µl of the culture

Members there:

• Instructors and advisors: Solenne.
• Students: Arnaud, Fabio, Juliette, Mathieu, Marie, Pierre, Romain, Sean and Terry.

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