Team:Paris Saclay/Notebook/July/8

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Notebook : July 8

Summary:

FNR regulator system:

  • performed another digestion for AmilCP, Pfnr plus sequence LacZ with Not I, Xho I, EcoR I and PST I.
  • electrophoresis for those digest products
  • enrichment culture for clone of the briobrick BBa_K1155000
  • electrophoresis for extracted products of last friday

Lab work


The electrophoresis (135 V for 30 minutes) for digest products were migrated in agarose gel (0.5X).

Results:

PS080713dig.jpg
  • Well 1,12 : marker
  • Well 2: AmilCP1 + Ecor I
  • Well 3: AmilCP2 + Ecor I
  • Well 4: AmilCp1 + Not I
  • Well 5: AmilCP2 + Not I
  • Well 6: AmilCp1 + Xho I
  • Well 7: AmilCp1 + Xho I
  • Well 8: LacZ 1 + Ecor I + Pst I
  • Well 9: LacZ 2 + Ecor I + Pst I
  • Well 10: Nothing
  • Well 11: fnr promoter
  • gel 0.8%


Overnight incubation at 37°C with agitation.
The electrophoresis for the plasmids which we had extracted last week plus 3 other BioBrick promoter Bph R1, promoter Bph A1, Bph R2.

Volume added:

  • Pfnr2: 10µl
  • R1P: 10µl
  • R2: 10µl
  • A1P: 10µl


Migration at 100V for 25mins then continued till 45 minutes:
Results:

PS08071345.jpg
  • Well 1 : R2
  • Well 2 : R1P
  • Well 3 : A1P
  • Well 4 : Pfnr
  • gel 0.8%


The concentration of Pfnr2 was detected by NanoDrop :

  • C = 1788.8 ng/µl
  • 260/280 = 2.08

We considered that our extraction of plasmid DNA was not successful, we need to perform another extraction DNA.








Electrophoresis band size estimation

We used Clonemanager for band size estimation:


Molecule enzymes(Buffer) Size
AmilCP clone Nod I(Orange) 2046+693bp
AmilCP clone XHOI(Red) 1842+892bp
BBa_K1155000 XHOI(Red) 1289+892bp
BBa_K1155000 ECoR I(Orange) 2151bp









Navigation Home Team Project Labwork Notebook Human practices

Notebook : August 23

Lab work

A - Aerobic/Anaerobic regulation system

Objective : Bba_K1155003, Bba_K1155007

1 - Digestion of Bba_K592009, Bba_K1155007 by NotI, XhoI, EcoRI, EcoRI/PstI

Abou, Anaïs, Sheng, Zhou

Used quantities :

  • NotI :
    • Bba_K592009 : 2µL
    • Buffer orange : 2µL
    • NotI : 0.5µL
    • H2O : 15.5µL
  • XhoI :
    • Bba_K592009 : 2µL
    • Buffer red : 2µL
    • XhoI : 0.5µL
    • H2O : 15.5µL
  • EcoRI :
    • Bba_K592009 : 2µL
    • Buffer orange : 2µL
    • EcoRI : 0.5µL
    • H2O : 15.5µL
  • EcoRI/PstI :
    • Bba_I732017 : 2µL
    • Buffer orange : 2µL
    • EcoRI : 0.5µL
    • PstI : 0.5µL
    • H2O : 15µL

We let our digestion 1h30 at 37°C.

2 - Electrophoresis of the digestion of Bba_K592009, Bba_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI

Abdou, Anaïs, Sheng, Zhou

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL Bba_K592009 clone 1 digested by EcoRI+1µl of 6X loading dye
  • Well 3 : 5µL Bba_K592009 clone 2 digested by EcoRI+1µl of 6X loading dye
  • Well 4 : 5µL Bba_K592009 clone 1 digested by NotI+1µl of 6X loading dye
  • Well 5 : 5µL Bba_K592009 clone 2 digested by NotI+1µl of 6X loading dye
  • Well 6 : 5µL Bba_K592009 clone 1 digested by XhoI+1µl of 6X loading dye
  • Well 7 : 5µL Bba_K592009 clone 2 digested by XhoI+1µl of 6X loading dye
  • Well 8 : 5µL Bba_I732017 clone 1 digested by EcoRI/PstI+1µl of 6X loading dye
  • Well 9 : 5µL Bba_I732017 clone 2 digested by EcoRI/PstI+1µl of 6X loading dye
  • Well 10 : -
  • Well 11 : -
  • Well 12 : 6µL DNA Ladder
  • Gel : 1%
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