Team:Paris Saclay/Notebook/July/28

From 2014.igem.org

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====PCR====
====PCR====
''by Sean''
''by Sean''
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Cultures used: MG1655Z1 10I and MG1655 100I.
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[https://2014.igem.org/Team:Paris_Saclay/Protocols/PCR_for_bacterial_culture Protocol]
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<span style="color:red">Note: although we have 2 cultures to prepare, it is recommended to prepare 3 tubes because of probable uncertainties when measuring volumes.</span>
===C - Lemon scent===
===C - Lemon scent===

Revision as of 12:13, 29 July 2014

Contents

Monday 28th July

Lab work

A - The frame

Preparation of electrocompetent cells

by Arnaud & Romain

Strain used: E. coli MG1655Z1 and E. coli MG1655.

Protocol:

Two dilution of 200µl of bacterial culture MG1655Z1 and E. coli MG1655 in 15ml of LB at 30°C for each strain.

When the culture OD650 = 0,6:

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

Transformation of electrocompetent cells

by Arnaud & Romain

Strain used: E. coli MG1655Z1 and E. coli MG1655. Plasmid used: BT640 (code for a flipase for E. coli)

Make 2 électroporations in cold electroporation cuvettes:

  • A control cuvette(without DNA): 50µl of E. coli MG1655Z1 or E. coli MG1655.
  • A second cuvette: 50µl of E. coli MG1655Z1 or E. coli MG1655 culture + 1µl of BT640 plasmid.

Electroporation : 2500V, 132W, 40µF.

After that, add 1ml of cold LB in each cuvette and transfer in 2 tubes.

Spread on 8 dishes LB + Cm:

  • 20µl of control E. coli MG1655Z1 (without plasmid)
  • 50µl of transformed E. coli MG1655Z1 with BT340.
  • 100µl of transformed E. coli MG1655Z1 with BT340.
  • Transformed and concentrated E. coli MG1655Z1 with BT340.
  • 20µl of control E. coli MG1655 (without plasmid)
  • 50µl of transformed E. coli MG1655 with BT340.
  • 100µl of transformed E. coli MG1655 with BT340.
  • Transformed and concentrated E. coli MG1655Z1 with BT340.

Incubate for a night at 30°C.

PCR

by Sean

Cultures used: MG1655Z1 10I and MG1655 100I.

Protocol

Note: although we have 2 cultures to prepare, it is recommended to prepare 3 tubes because of probable uncertainties when measuring volumes.

C - Lemon scent

Liquid Culture

by Fabio

Due to an widespread infection of the cells made the 25th July, we collected the original strain DY330 to verify it's legitimacy.

2ml LB + 20μl of DY330. We incubate cultures at 30°C.

E - Salicylate Inducible Suppressing System

Plasmid DNA Purification

by Fabio

  • BBa_J61051
  • BBa_K228001

from Bacterial Culture made the 25th July.

Protocol

Digestion

by Fabio

Once we have a good amount of our BioBricks' plasmid, it's time to do the digestion.

TODO: Digestion's protocol

Electrophoresis

by Fabio

The Electrophoresis was used to verify the success of the Digestion and the success of the plasmid DNA purification at the same time.

  1. BBa_J61051 Cl.1
  2. BBa_J61051 Cl.2
  3. BBa_K228001 Cl.1
  4. BBa_K228001 Cl.2

Results:

(TODO: Photos from LU000101 to LU000105)

Members present:

  • Instructors and advisors: Solenne and Sylvie.
  • Students: Arnaud, Fabio, Romain, Sean and Terry.

Back to the calendar

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