Team:Paris Saclay/Notebook/July/30

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===C - Salicylate Inducible Suppressing System===
===C - Salicylate Inducible Suppressing System===
====DNA purification gel agarose====
====DNA purification gel agarose====
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''by Fabio''
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''In progress''
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Once the segregate process made [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/29#Electrophoresis yesterday] by electrophoresis, the DNA correspondent to the core of '''BBa_J61051''' was purified from the gel agarose.
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[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Segregate_process Segregate Process Protocol]
====Ligation====
====Ligation====
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''by Fabio''
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The final step to have a BioBrick Assembly is the Ligation reaction.
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<font color="#F00">TODO: illustration of the process</font>
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* BioBrick '''BBa_J61051''' (Salicylate promoter + NahR) as '''Part A'''
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* BioBrick '''BBa_K228001''' (RNA suppressor) as '''Part B'''
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''In progress''
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[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Ligation BioBrick Assembly - Ligation Protocol]
===D - Lemon scent===
===D - Lemon scent===

Revision as of 12:26, 1 August 2014

Contents

Wednesday 30th July

Lab Work

A - The frame coli Odor free

Preparation of electrocompetent cells

by Romain

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

Protocol:

Two dilution of 500µl 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 Romain

Strain used: E. coli MG1655Z1 and E. coli MG1655. Plasmid used: BT340 (code for a flipase for E. coli)

Make 2 electroporations 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.

Incubate during 1h at 30°C.

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.

C - Salicylate Inducible Suppressing System

DNA purification gel agarose

by Fabio

Once the segregate process made yesterday by electrophoresis, the DNA correspondent to the core of BBa_J61051 was purified from the gel agarose.

Segregate Process Protocol

Ligation

by Fabio

The final step to have a BioBrick Assembly is the Ligation reaction.

TODO: illustration of the process

  • BioBrick BBa_J61051 (Salicylate promoter + NahR) as Part A
  • BioBrick BBa_K228001 (RNA suppressor) as Part B

BioBrick Assembly - Ligation Protocol

D - Lemon scent

PCR of BBa_K762100

by Sean

This was a second attempt at yesterday's PCR, with several changes in parameters. Two tubes were prepared: one with yesterday's enzyme and another with a newer version of the same enzyme in order to determine if yesterday's enzyme was one of the causes of the rather unsatisfactory results.

Oligonucleotides used: iPS66, iPS67

Oligonucleotides were diluted twice from 100µM to 50µM.

Protocol

Add into each PCR tube the following:

Component For a total volume of 50μl
H2O 35.5μl
Phusion buffer 5X 10μl
dNTPs 1μl
iPS66 1μl
iPS67 1μl
BBa_K762100 1μl
Phusion DNA polymerase 0.25μl

Tube was placed in PCR machine with the following parameters.

Cycle step Temperature Time Cycle

Initial denaturation

98°C

1 min

1

Denaturation 98°C 15 s 25 - 30
Annealing 52°C 20 s 25 - 30
Extension 72°C 45 s 25-30
Final extension 72°C 10 min 1
Final extension 8°C hold 1

Members there:

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

Back to the calendar

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