Team:Paris Saclay/Notebook/August/5

From 2014.igem.org

(Difference between revisions)
(Tuesday 5th August)
(PCR verification)
Line 116: Line 116:
Results of the PCR: The electrophoresis revealed correctly the expected size of the sequence, except for the holes 2 and 11.
Results of the PCR: The electrophoresis revealed correctly the expected size of the sequence, except for the holes 2 and 11.
 +
The kan resistance sequence had been successfully removed.
===C - Salicylate Inducible Suppressing System===
===C - Salicylate Inducible Suppressing System===

Revision as of 08:14, 6 August 2014

Contents

Tuesday 5th August

Lab work

A - The chassis coli Odor free

PCR verification

by Romain

Results of the striates on dishes from yesterday:

For the strain MG1655Z1:

  • Nothing has grown on the kan dish, but the colony grew on LB dish. That was what we expected, the kan cassette was correctly delete.

For the strain MG1655:

  • Few colonies have grown on the kan dish, and all the colonies have grown on LB dish. It's a little failure.

So now, we proceed to the PCR to verify the length of the sequences.

Strains used: E. coli MG1655Z1 and E. coli MG1655, and oligonucleotides used: iPS75 bis and iPS76 bis.

Protocol

Add into a PCR tube the following:

Component For a total volume of 50μl
H2O 28.25μl
Green GoTaq buffer 5X 10μl
dNTPs 1μl
iPS75 bis 2μl
iPS76 bis 2μl
DMSO 1.5μl
MgCl2 4μl
Bacterial culture 2μl
Green GoTaq enzyme 0.25μl

We follow these quantities for 11 tubes: 5 tubes for the strain MG1655, 5 tubes for the strain MG1655Z1 and 1 tube with bacteria which had grown on the kan dish. Tube was placed in PCR machine with the following parameters.

Cycle step Temperature Time Cycle

Initial denaturation

95°C

10 min

1

Denaturation 95°C 30 s 30
Annealing 53°C 30 s 30
Extension 72°C 2 min 30
Final extension 72°C 5 min 1
Final extension 12°C hold 1

The different bacterial cultures in each tubes:

  1. Tube 1 to 5: MG1655
  2. Tube 6 to 10: MG1655Z1
  3. Tube 11: MG1655 which had grown on the kan dish.
050814-Romain-PCRa.jpg

The electrophoresis results:

1. 10 µl of ladder
2 to 6. 10 µl in each tube of PCR for the strain MG1655
7 to 11. 10 µl in each tube of PCR for the strain MG1655Z1
12. 10µl of PCR for the MG1655 which had grown on the kan dish

Results of the PCR: The electrophoresis revealed correctly the expected size of the sequence, except for the holes 2 and 11. The kan resistance sequence had been successfully removed.

C - Salicylate Inducible Suppressing System

Liquid Culture

by Fabio

2 liquid cultures of BBa_K1372000 with 20ml of LB and 20µl of amp (at 3pm - 150 rpm - 37 °C), from the stock made the 4st August.

D - Lemon scent

Electrophoresis

050814-Sean-Pierre-PCRa.jpg

by Sean & Pierre The strains used were made the 4th August

  1. 5 µl of BBa_K762100 non diluted
  2. 5 µl of BBa_K762100 diluted 10-1
  3. 5 µl of BBa_K517003

Protocol

Human Pratices

by Pierre

Ethics

I wrote the begining of a global view of the evolution of the definitions of life from Aristotle to modern times.

Bibliography

  • Aristotle, On the Soul
  • Epicurus, Letter to Herodotus
  • Descartes, Discourse on the method
  • Darwin, On the origin of species
  • Bichat, Physiological researches on life and death

by Hoang Vu

Continue my bibliographical research on Citral A and B and Beta-Pinene. I have now a little more than twenty reviews about various uses and properties of citral


Members there:

  • Instructors and advisors: Alice and Sylvie.
  • Students: Eugene, Fabio, Hoang Vu, Leila, Juliette, Pierre, Romain, Sean and Terry.

Back to the calendar