Team:Paris Saclay/Notebook/July/22

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*1ml of bacterial culture (250µl on each dish)
*1ml of bacterial culture (250µl on each dish)
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Results: [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/24 24th July]
'''Members there''':
'''Members there''':

Revision as of 10:11, 24 July 2014

Contents

Tuesday 22nd 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: (For each clone)

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
  • 1ml of bacterial culture (250µl on each dish)

Results: 24th July

Members there:

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

Back to the calendar

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Notebook/July/22"