Notebook

• Week

  25

  NOTE TITLE

  • Jun

    18

    Note title

    Test.
  • Jun

    19

    Note title

    Lorem ipsum dolor sit amet.
  • 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

    Note title

    Lorem ipsum dolor sit amet.
  • Jun

    26

    Note title

    Lorem ipsum dolor sit amet.
  • Jun

    27

    Note title

    Lorem ipsum dolor sit amet.
  • Jun

    28

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  • 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

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  • Jul

    9

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  • Jul

    10

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  • Jul

    11

    Note title

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  • Jul

    12

    Note title

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  • Jul

    13

    Note title

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• Week

  29

  Note title

  • Jul

    16

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  • Jul

    17

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  • Jul

    18

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  • Jul

    19

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  • Jul

    20

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• Week

  30

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  • Jul

    23

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  • Jul

    24

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  • Jul

    25

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  • Jul

    26

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  • Jul

    27

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• Week

  31

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  • Jul

    30

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  • Jul

    31

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  • Aug

    1

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  • Aug

    2

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  • Aug

    3

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• Week

  32

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  • Aug

    6

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  • Aug

    7

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  • Aug

    8

    Note title

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  • Aug

    9

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  • Aug

    10

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• Week

  33

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  • Aug

    13

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  • Aug

    14

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  • Aug

    15

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  • Aug

    16

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  • Aug

    17

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• Week

  34

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  • Aug

    20

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  • Aug

    21

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  • Aug

    22

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  • Aug

    23

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  • Aug

    24

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