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.
    
    Results:
    Sett inn bilde her.
  • Jun

    27

    Note title

    Lorem ipsum dolor sit amet.
  • Jun

    28

    Note title

    Lorem ipsum dolor sit amet.
  • Jun

    29

    Note title

    Lorem ipsum dolor sit amet.
• Week

  27

  Note title

  • Jul

    1

    Note title
  • Jul

    2

    InterLabs Study

    Calibration 1 and 2 for the InterLabs Study.
  • Jul

    3

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    4

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    5

    Note title

    Lorem ipsum dolor sit amet.
• Week

  28

  Note title

  • Jul

    9

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    10

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    11

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    12

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    13

    Note title

    Lorem ipsum dolor sit amet.
• Week

  29

  Note title

  • Jul

    16

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    17

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    18

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    19

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    20

    Note title

    Lorem ipsum dolor sit amet.
• Week

  30

  Note title

  • Jul

    23

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    24

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    25

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    26

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    27

    Note title

    Lorem ipsum dolor sit amet.
• Week

  31

  Note title

  • Jul

    30

    Note title

    Lorem ipsum dolor sit amet.
  • Jul

    31

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    1

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    2

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    3

    Note title

    Lorem ipsum dolor sit amet.
• Week

  32

  Note title

  • Aug

    6

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    7

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    8

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    9

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    10

    Note title

    Lorem ipsum dolor sit amet.
• Week

  33

  Note title

  • Aug

    13

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    14

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    15

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    16

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    17

    Note title

    Lorem ipsum dolor sit amet.
• Week

  34

  Note title

  • Aug

    20

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    21

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    22

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    23

    Note title

    Lorem ipsum dolor sit amet.
  • Aug

    24

    Note title

    Lorem ipsum dolor sit amet.