Team:Pasteur Paris/Week 12

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Week 12

08/17 - 08/21




Gibson Assembly


    NB -Esterase assay

      Gel Purification


Tubes

Ladder

1st transformation

 

Components

 

GeneRuler 1kb DNA Ladder
(6x DNA loading dye)
3µL

Buffer
4µL
+
DNA
20µL
    The bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3.
    - Bacterial culture of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well.
    - Midi-prep has been done with the 50mL of BBa_J61047 culture. (dilution 1/1000)

     

    DNA Concentration (ng/µl)

    OD(260nm)

    OD(280nm)

    OD(260nm)/OD(280nm)

    BBa_J61047

    0,5

    0,011

    0,004

    2,57

    • tube 1 « Miniprep XbaI/SpeI » : 5uL 10X buffer cutsmart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.
    • tube 2 « Midiprep XbaI/SpeI » : 5uL 10X buffer cut smart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour
    • tube 3 « Miniprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
    • tube 4 « MiDiprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
    • tube 5 « Miniprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.
    • tube 6 « Midiprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.
    • MWT : « Quick-Load Purple 2-Log DNA Ladder (0.1-10.0 kb) NEB

      NB-Esterase Assay:

         

        Concentration (ng/µL)

        OD(230nm)

        OD(260nm)

        OD(280nm)

        OD(260nm)/OD(230nm)

        OD(260nm)/OD(280nm)

        Cluster_4_start

        30

        0,025

        0,575

        0,375

        1,50

        -

        /936011

        55

        0,4

        1,075

        0,575

        1,88

        2,65

        936011/936023

        7,5

        0,6

        0,125

        0,05

        2,41

        0,21

        936023/

        45

        0,575

        0,925

        0,625

        1,48

        1,61

        Cluster_4_end

        47,5

        0

        0,925

        0,575

        1,64

        -

^
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