Team:NTNU Trondheim/Experiments

Lab Journal


  • Week
    25

    Getting started

    • Jun
      18

      No lab

      No lab.
    • Jun
      19

      No lab

      No lab.
    • Jun
      20

      Our first day in the laboratory!

      Goal: Incubate the Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 on agar plates with antibiotics.

      Preparation of LA- medium
      LA- medium (1.5%) were made after the following recipe, and autoclaved for 20min at 121°C.
      • 1L Distilled water
      • 5g Yeast extract
      • 10g Tryptone
      • 5g NaCl
      • 15g Agar

      Agar plates with antibiotics
      Two different agar plates were made – one type contains ampicillin (AMP) while the other type contains chloramphenicol (CM), and were prepared after the following recipe:

      LA- medium with AMP:
      • 0.5L LA-medium
      • 0.5mL AMP (50mg/mL)
      LA-medium with CM:
      • 0.5L LA-medium
      • 0.5mL CM (35mg/mL)

      The LA-media with antibiotic were poured on petri dishes.

      Incubation of bacteria
      Two Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 plasmids respectively, were ordered from Addgene. E.coli which carry the pgRNA plasmid were plated on agar plates with AMP, while E.coli which carry the pdCas9 plasmid were plated on two agar plates with CM, and incubated at 37℃.

      Results:
      The E.coli with pgRNA and pdCas9 formed colonies on the agar plates with AMP and CM
    • Jun
      21

      Inoculation of E.coli

      Goal: Inoculate a colony from each agar plate which were prepared from the previous day, in LB- medium with antibiotics.

      Procedure
      One colony of pgRNA- and pdCas9- bacteria grown overnight on agar plates were picked and inoculated in LB-media (25mL) with AMP (25μL) and CM (25μL), respectively. The cell cultures were incubated at 37℃ in a shaking incubator at 204rpm.
    • Jun
      22

      Isolation and verification of pgRNA and pdCas9

      Goal: Isolate and verify that the E.coli carry pgRNA and pdCas9.

      Plasmid isolation
      pgRNA and pdCas9 were isolated from the incubated bacteria prepared yesterday (June 21) by following the protocol of ZR Plasmid Miniprep Kit:
      1. 1mL of each cell culture (which either carry pgRNA or pdCas9) were transferred to eppendorf tube and centrifugated for 20s at 16000g
      2. The supernatant was discarded, while the pellet was resuspended in buffer
      3. Lysing buffer was added to the sample and carefully mixed in 2min
      4. Neutralizing buffer was added to the sample, and mixed until the colour of the solution became yellow
      5. The solution was centrifugated for 2min at 16000g
      6. The supernatant was collected and transferred to column in a collection tube, and centrifugated for 30s at 16000g
      7. The supernatant in the collection tube was discarded
      8. Endo-wash buffer was added to the column and centrifugated for 1min at 16000g
      9. Plasmid- wash buffer was added to the column and centrifugated for 1 min at 16000g
      10. The solution in the collection tube was discarded, while the column with plasmids was transferred to a new collection tube
      11. The plasmids in the column were eluted by adding 30μL distilled water, and centrifugated for 30s in 16000g

      Determine plasmid concentration and purity
      The concentration of plasmids and the purity of each sample were determined by using Nanodrop.

      Restriction digest and plasmid verification
      The presence of pgRNA and pdCas9 in the cell cultures were verified after following the restriction digest protocol and separation of the DNA fragments by gel electrophoresis.
  • Week
    26

    Note title

    • Jun
      25

      Transformation of pgRNA and pdCas9 into competent cells

      Goal: Transform pgRNA and pdCas9 whinto competent cells (E.coli K-12 DH5α).

      Procedure:
      pgRNA and pdCas9 which were isolated from day 3 (22. June. 2018) were transformed into E.coli K-12 DH5α cells after the following transformation protocol:
      1. Thaw competent cells on ice for 10min.
      2. Use a pipette and transfer 2μL of mini-prepped plasmids into DH5α samples. Pipette up and down while stirring the pipette tip gently for 5-10s.
      3. Incubate the cells on ice for 20min.
      4. Heat shock the cells for 45s at 42℃. Immediately put the samples on ice for 2min.
      5. Add 900μL LB medium to the cells. Incubate at 37℃, 200RPM for 1h.
      6. Plate out 100μL of the transformation medium into agar plates with selection agent (in our case, we chose chloramphenicol).
      7. Incubate the cells at 37℃.

      Procedure:
      Sett inn bilder.
    • Jun
      26

      Transfer successful transformed cells to LB-medium and preparation of standard curves for interLab study

      Goal:
      Inoculate successful transformed competent cells to LB-medium, and prepare the standard curves for interLab study.

      Procedure:
      Inoculate colony from agar plates to LB-medium:
      A colony from each agar plate were selected and inoculated to LB- medium with antibiotics. Competent cells with pgRNA were transferred to LB- medium with AMP, while competent cells with pdCas9 were inoculated into LB- medium with CM. The cell cultures were incubated at 37℃ with shaker.

      Preperation of 1xPBS splution:
      1xPBS solution for the interLab study was made after the following recipe:
      • 1L Distilled water
      • 8g NaCl
      • 0.2g KCl
      • 1.42g Na2HPO4
      • 0.24g KH2PO4

        • Prepareation of agar plates, LA- and LB-medium:
        LA- and LB- medium was made after the protocols from day 1 (20. June. 2018).
        Agar plates with CM were prepared after the protocol from day 1(20. June. 2018).

        Preparation of standard curves for interLab study:
        Calibration 1: OD600 reference point – LUDOX protocol
        A conversion factor to transform absorbance data from plate reader into OD600 was obtained in spectrometer by measuring 4 replicates of LUDOX CL-X and dd H2O. The data were imported into Excel sheet.
        Calibration 2: Particle standard curve – Microsphere protocol
        Solution of silica beads provided in kit from iGEM HQ, was vortexed before dilution with water (96μL Silica beads in 904μL dd H2O). A serial dilution of microspheres was obtained by adding 100μL dd H2O into 4x11 wells (E2, F2, G2, H2…E12, F12, G12, H12) on a 96 well plate. Microsphere stock solution (200μL) was pipetted into E1 and 100μL of the solution was transferred to E2 and mixed well before transferring 100μL to E3. The procedure repeated until 100μL solution was pipetted into E11, and 100μL was transferred to liquid waste. The dilution series was repeated for row F, G and H. The absorbance of the samples was measured by a plate reader with shaker at 24℃. The data were exported to Excel sheet.
    • Jun
      27

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      28

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      29

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  • Week
    27

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    • Jul
      1

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    • Jul
      2

      InterLabs Study

      Calibration 1 and 2 for the InterLabs Study.
    • Jul
      3

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      4

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      5

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  • Week
    28

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      10

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      11

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      12

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    29

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      24

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      25

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      26

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    31

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      31

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      1

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    33

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      16

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      17

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  • Week
    34

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      20

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      21

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      22

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      23

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      24

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