Experiments

Main steps：

  • PCR
  • Agarose gel electrophoresis | Target DNA fragment extraction
  • LB culture medium

Details：

1.1. Experiment materials：

  1. 1) Tris-Acetate-EDTA buffer
  2. 2) Buffer GW
  3. 3) GDP
  4. 4) ddH2O
  5. 5) 5xPS Buffer
  6. 6) dNTPs
  7. 7) primer F： FoxO1-forward
  8. 8) primer R： FoxO1-reverse
  9. 9) Template DNA
  10. 10) PrimeStar HS polymerase
  11. 11) Nucleic acid dye
  12. 12) Agarose
  13. 13) DNA marker
  14. 14) LB culture powder

1.2. Experiment devices：

  1. 1) PCR instrument
  2. 2) Pipettes
  3. 3) Centrifuge tube
  4. 4) Microwave oven
  5. 5) Volumetric flask

1.3. Experiment procedures：

  1. 1.3.1) PCR
    1. a) PCR reaction system
      1. 11.5ul ddH2O
      2. 5ul 5xPS buffer
      3. 2ul dNTPs
      4. 2.5ul F-FoxO1 forward
      5. 2.5ul R-FoxO1 reverse
      6. 1ul Template DNA
      7. 0.5ul Prime STAR
    2. b) PCR program
        Put microcentrifuge tube in PCR instrument
      1. 98°C pre-denature 5min
      2. 98°C denature 30s
      3. 52°C annealing 30s
      4. 72°C extension 2min
        1. iii-v step repeats 30 times
        2. amplicons keep at 4°C before use
  2. 1.3.2) Agarose gel electrophoresis
    1. a) Prepare agarose gel
      1. 100ml 1xTAE buffer + 1.5g agarose
      2. Melt the agarose with a microwave oven
    2. b) Gel electrophoresis
      1. Put the molten agarose into a gel box
      2. Put agarose gel into electrophoresis tank after molten agarose is solidified
      3. Dye the DNA with Nucleic acid dye
        1. Add 2.5ul DNA dye to each sample
      4. Add samples to the wells in the agarose gel
        1. Stay a well for adding DNA marker
      5. Turn on the electrophoresis tank
        1. Electrophoresis tank set at 120v，20min
      6. Put the agarose gel on the UV Transilluminator
        1. Find the correct size range band, cut it off, and put it into a 1.5ml centrifuge tube
    3. c) Gel Extraction
      1. Add 300ul buffer GDP into each 1.5ml microcentrifuge tube
      2. heat in a thermostat at 42°C for 5min
      3. Transfer the sample solution from the 1.5ml centrifuge tube into a new 2ml collection tube (do have a filter column in the 2ml collection tube)
      4. Put the 2ml collection tube into the laboratory microcentrifuge, centrifuge and discard the liquid
        1. 2min，12000rpm x2
      5. Add 700ul buffer GW to each column
        1. 1min，12000rpm x2
      6. Discard liquid and centrifuge the empty tube again
        1. 2min，12000rpm
      7. Place the column into a new 1.5ml microcentrifuge tube, and add 30ul ddH2O directly to the center of the column matrix. Centrifuge to elute the DNA fragment
        1. 2min，12000rpm
  3. 1.3.3) Configuration LB culture medium
    1. Add LB culture powder to a 1L volumetric flask
      1. 200ml purified water, 5g LB culture powder
      2. 100kPa, 120°C, 20min

Main steps：

  • Exchange cell culture medium
  • Count cell number
  • Bacteria culture

Details：

2.1. Experiment materials：

  1. 1) Fetal bovine serum (FBS)
  2. 2) Minimum Essential Medium (MEM medium)
  3. 3) Pancreatin solution
  4. 4) Penicillin-streptomycin solution
  5. 5) HepG2 cell

2.2. Experiment devices：

  1. 1) Pipettes
  2. 2) 48-well plate
  3. 3) Cell Viability analyzer system
  4. 4) Cell culture flask
  5. 5) Culture plate
  6. 6) Centrifuge tube
  7. 7) Incubator

2.3. Experiment procedures：

  1. 2.3.1) HepG2 Cell culturing & number count
    1. a) Cell culturing
      1. Configuration PBS mixed solution
        1. 45ml PBS + 5ml pancreatin
      2. Take off the original culture medium (MEM)
      3. Add PBS mixed solution and discard the liquid
      4. Add 1ml pancreatin solution to dissociation cell for 2min
      5. Resuspend the cell solution and transfer it to a corresponding 50ml centrifuge tube
      6. Centrifuge the solution and discard the supernatant
        1. 1. 1000rpm, 5min
      7. Add 5ml new culture medium to a new 6ml cell culture flask
        1. 5ml new culture medium (4ml original culture medium + 1ml FBS)
      8. Resuspend cell solution and transfer it to the cell culture flask
      9. Shake gently and culture it in the incubator
        1. 37°C, 5% CO2
    2. b) Exchange cell culture medium and culturing
      1. Configuration cell culture medium
        1. 5ml FBS + 45ml MEM + 0.5ml p/s
      2. Suck the original culture medium by pipette
      3. Add 5ml PBS to cover the cell
        1. Repeat those steps two times
      4. Add 2ml pancreatin solution
        1. Shake gently
      5. Culture the cell in the incubator
        1. 37°C, 2min
      6. Add 3ml MEM&FBS mixed solution into the cell culture flask
        1. Resuspend the cell
    3. c) Cell counter
      1. Take 200ul cell solution and put it into the Cell Viability analyzer system
        1. 0.79*106 cells/ml
      2. Add mixed solution (1.5ml cell solution + 10ml MEM&FBS culture medium) to the 48 well plate
        1. Each well add 0.5ml culture medium
  2. 2.3.2) Bacteria culture
    1. a) inoculate the E.coli to volumetric flask that contain LB culture medium
      1. 37℃, 220rpm

Main steps：

  • PCR
  • T4 DNA Digestion
  • Cell transfection with LipoFiter 3.0

Details：

3.1. Experiment materials：

  1. 1) ddH2O
  2. 2) 5xPS Buffer
  3. 3) dNTPs
  4. 4) F：FoxO1-forward
  5. 5) R：FoxO1-reverse
  6. 6) Template DNA
  7. 7) PrimeSTAR
  8. 8) pcDNA3.1-hFoxO1 plasmid
  9. 9) FD green buffer
  10. 10) XhoI / KpnI
  11. 11) T4 DNA Ligase buffer
  12. 12) T4 DNA Ligase
  13. 13) Nuclease-free water
  14. 14) HepG2 cells
  15. 15) Cell culture medium
  16. 16) LipoFiter 3 Liposomal transfection reagent
  17. 17) Serum
  18. 18) DMEM solution

3.2. Experiment devices：

  1. 1) PCR instrument
  2. 2) Pipettes
  3. 3) microcentrifuge tube
  4. 4) cell incubator
  5. 5) serum-free medium
  6. 6) medium with serum

3.3. Experiment procedure：

  1. 3.3.1） PCR
    1. a) PCR reaction system
      1. 13.9ul ddH2O
      2. 5ul 5xPS buffer
      3. 2ul dNTPs
      4. 1.3ul F-FoxO1 forward
      5. 1.3ul R-FoxO1 reverse
      6. 1ul Template DNA
      7. 0.5ul Prime STAR
    2. b) PCR program
        Put centrifuge tube in PCR instrument
      1. 98°C pre-denature 5min
      2. 98°C denature 30s
      3. 52°C annealing 30s
      4. 72°C extension 5min
        1. iii-v step repeats 30 times
        2. amplicons keep at 4°C before use
  2. 3.3.2) T4 DNA Ligase
    1. a) 200ul microcentrifuge tube
        Add to the same microcentrifuge tube after PCR program
      1. 4ul pcDNA3.1
      2. 5ul FD green buffer 37°C > 4h
      3. 2ul XhoI
      4. 2ul KpnI
      5. 3ul ddH2O
  3. 3.3.3) LipoFiter 3 Liposomal transfection reagent
    1. Cultivate HepG2 cells
    2. Replace the culture medium in the well of the 6-well plate
      1. a) add 2ml fresh cell culture medium.
    3. Take a new centrifuge tube to mix the following reagents:
      1. a) 4ug of plasmid DNA
      2. b) 260 ul DMEM solution
    4. Take another centrifuge tube to mix the following reagents:
      1. a) 250ul DMEM solution
      2. b) 6 ul LipoFIlter 3
      3. c) Place the mixture at room temperature for 5 min
    5. Mix the DNA mix solution and LipoFilter 3 in step 4 and 5
    6. Add 500 ul LipoFilter 3-DNA mixture into each well of the 6-well plate
    7. After cultivating the cells in the cell incubator for 6-12 hours,
      1. a) Remove the serum-free medium containing LipoFilter 3-DNA.
      2. b) Add 2ml of fresh cell culture medium with serum for further cultivation of the cells.

Main steps：

  • Western blot
  • Dual Luciferase report assay
  • Glucose & protein assay

Details：

4.1. Experimental materials：

  1. 1) PBS buffer
  2. 2) Cell lysis buffer
  3. 3) Firefly Luciferase
  4. 4) Renilla Luciferase
  5. 5) Luciferase assay buffer II
  6. 6) Luciferase Assay Substrates
  7. 7) Western blot kit
  8. 8) PVDF membrane
  9. 9) 1x TBS buffer
  10. 10) PBST buffer
  11. 11) FoxO1 antibody

4.2. Experimental apparatuses：

  1. 1) 10ul pipettes
  2. 2) 96-well cell plates
  3. 3) microplate shaker
  4. 4) microplate reader
  5. 5) electrophoresis chamber
  6. 6) shaking platform

4.3. Experimental procedures：

  1. 4.3.1 Western blot
    1. SDS-PAGE gel electrophoresis
    2. Once the gel electrophoresis is completed, remove the gel from the electrophoresis chamber, and cut the stacking gel.
    3. Immerse the gel in the transfer buffer for 20 minutes; immerse the membrane and the filter papers in the transfer buffer. Place one holder with a sponge, 3 sheets of filter paper. Dry the membrane and place in the dark.
    4. Place the sandwich in the transfer chamber, filled with transfer buffer, make sure that the membrane is facing the red electrode. Run the transfer according to the membrane area and protein MW.
    5. Place the membrane in a heat sealable bag; add block solution 0.1-0.2 mL for each square centimeter of membrane; squeeze out air as much as possible; seal the bag, place on a shaking platform for 1 hour at RT.
    6. Add block solution 0.1-0.2 mL for each square centimeter of membrane; add antibody, squeeze as much air as possible from the bag; seal the bag, and place on a shaking platform for 1 hour at RT.
    7. Wash membranes in PBST (0.05% Tween in PBS) on a shaking platform for 5 minutes. Repeat 3 times.
    8. Wash the membrane with TBS for 10 min; add block solution 0.2 mL; add HRP -secondary antibody, squeeze as much air as possible from the bag; seal the bag, place on a shaking platform for 1 hour at RT.
    9. Wash membrane in TBST (0.05% Tween) on a shaking platform for 5 minutes. Repeat 3 times total; Wash again in TBS.
    10. Stop reaction after the expected band appears by pouring out the substrate and rinsing with distilled water repeatedly. Dry the membrane and place in the dark.
  2. 4.3.2 Luciferase report assay
    1. a) Luciferase experiment procedures
      1. Remove supernatant from filter tube and wash with PBS
        1. remove culture medium and wash with PBS for two times
      2. Add cell lysis buffer
        1. the ratio of lysate and PBS used is：1ml cell culture +4ml PBS solution
        2. add 80ul of lysis buffer to each sample
        3. Place in microplate shaker for 20min
      3. Add 20ul lysate into Luciferase 96-well plate
      4. detect fluorescence intensity with luciferase assays
        1. Prepare Firefly Luciferase
        2. Prepare Renilla Luciferase
        3. Configure Luciferase Assay Substrate（LAR II/Luciferase assay buffer II + Luciferase assay substrate）
        4. Add 100ul of Luciferase Assay Substrate to each sample in the 96-well plate a) resuspend 2-3 times
        5. Add 100ul of Stop & Glo○R Reagent to each sample in the 96-well plate
          The below Table shows the configuration of the 24 samples （keeping only 24 wells with samples）
          Sample 1 Sample 1a Sample 2 Sample 2a
          Sample 3 Sample 3a Sample 4 Sample 4a
          Sample 5 Sample 5a Sample 6 Sample 6a
          Sample 7 Sample 7a Sample 8 Sample 8a
          Sample 9 Sample 9a Sample 10 Sample 10a
          Note： Sample X a is a copy of the corresponding number sample
        6. Add dual-luciferase (Firefly Luciferase & Renilla Luciferase) into the samples
        7. Place the 96-well plate into the microplate reader and test for the fluorescence value
        8. Use 550-570nm wavelength to detect the intensity of Firefly luciferase
        9. Use 480nm wavelength to detect the fluorescence value of Renilla luciferase；
    2. b) Data analysis
      1. Collect data of the corresponding wavelength’s fluorescence intensity
        1. Calculate the average fluorescence intensity of the original samples
      2. Calculate the ratio of luciferin intensity in each well
        1. Firefly Luciferase fluorescence intensity was divided by the fluorescence intensity of Renilla Luciferase
      3. Create graphs according to the relevant data
    3. c) Analysis of common problems
      1. The test for reporter gene may be affected by the following circumstances: vector concentration、cell growth state、transfection efficiency、lysis efficiency、accuracy of samples used、detection procedure etc. All the above circumstance would result in an amount of variation on accuracy.
  3. 4.3.3 Glucose & protein assay
    1. a) Glucose & protein experiment procedures
      1. 50ul cell lysate +100ul reducing sugar detection reagent (24 samples)
      2. 20ul cell lysate +200ul modified Bradford reagent(24 samples)
      3. Place the 96-well plate into the microplate reader and test for the Glucose & protein concentration value
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