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Wet Lab Notes Part I
Experiment topic: polyacrylamide gel electrophoresis validation experiment

Experiment Period：2024.5.1-2024.5.15

Experimenters: Ruiying Wang, Lanxin Huang

Experiment Purpose:

To verify the feasibility of target participation in DDSD self-enhancement system.

Experimental Procedures:

(1) Sample preparation (20 uL)

Experimental group 1Report probe 1 uL, DDSD 1 uL, buffer2 uL, Bst 1 uL, target 1 uL, Rep chain 1 uL, water 13 uL
Control 1Report Probe 1 uL, DDSD 1 uL, Buffer 2 uL, Bst 1 uL, Rep Chain 1 uL, Water 13 uL
Control 2Report Probe 1 uL, Water 19 uL
Control 3DDSD 1 uL, Water 19 uL
Control 4Rep Chain 1 uL, Water 19 uL
Control 5Fluorescent Chain F 1 uL, Water 19 uL

(2) Electrophoresis

  1. Wash the glass plate, install the gel holder and check the leakage, and shake off the water for use. 2.
  2. Add the following components to the conical flask, shake well promptly after addition, pour into the gel holder, insert the comb, and leave to solidify.

    3. 12% Native Page(10 ml).

    ultrapure water5 mL
    40% Acrylamide3 mL
    5*TBE2 mL
    10%APS55 μL
    TEMED7 μL

    18% Native Page(10 ml).

    Ultrapure Water3.5 mL
    40% Acrylamide4.5 mL
    5*TBE2 mL
    10%APS55 μL
    TEMED7 μL

  3. Add 2 μL of 6x loading buffer to the EP tube; then take 5 μL of sample and add it to the EP tube separately.
  4. Pour appropriate amount of 0.5x TBE (no Mg2+) into the electrophoresis tank, take 5 μL of sample and marker and add it to the solidified gel, set the voltage at 120 V and run the gel for 60 min.
  5. Take 2.5 uL of dye, 25 ml of 0.5x TBE and add it into the plastic tank, remove the gel block and put it into the dye, shake the bed for about 15 min.
  6. Observe the result, open the instrument and software, wipe the sample stage with alcohol, put the result on the sample stage, select the blue light to observe the result and save it. Remove the sample, wipe the sample with alcohol and switch off the instrument.

    7. Experiment result:


    Experiment Topic: polyacrylamide gel electrophoresis validation experiment

    Experiment Period：2024.5.15-2024.5.30

    Experimenters: Ruiying Wang, Lanxin Huang

    Purpose of the experiment: to verify the necessity of Cas12 A protein to participate in the cristae reaction

    Experimental Procedures:

    (3) Sample preparation (20 uL)

    Experimental group 1Report probe 1 uL, target 1 uL, Cas12 A protein 1 uL, buffer 2 uL, crRNA 2 uL, water 13 uL
    Control 1Report probe 1 uL, target 1 uL, buffer 2 uL, crRNA 2 uL, water 14 uL
    Control 2Report probe 1 uL, target 1 uL, buffer 2 uL, crRNA 2 uL, water 19 uL
    Control 3target 1 uL, water 19 uL

    (4) Electrophoresis

  7. Wash the glass plate, install the gel holder, check for leaks, and shake out the water.
  8. Add the following components to the conical flask, shake well promptly after addition, pour into the gel holder, insert the comb and leave to solidify.

    9. 12% Native Page(10 ml).

    ultrapure water5 mL
    40% Acrylamide3 mL
    5*TBE2 mL
    10%APS55 μL
    TEMED7 μL

    18% Native Page(10 ml).

    Ultrapure Water3.5 mL
    40% Acrylamide4.5 mL
    5*TBE2 mL
    10%APS55 μL
    TEMED7 μL

  9. Add 2 μL of 6x loading buffer to the EP tube; then take 5 μL of sample and add it to the EP tube separately.
  10. Pour an appropriate amount of 0.5x TBE (no Mg2+) into the electrophoresis tank, take 5 μL of sample and marker and add it to the solidified gel, set the voltage at 120 V and run the gel for 60 min.
  11. Take 2.5 uL of dye, 25 ml of 0.5x TBE and add it into the plastic tank, remove the gel block and put it into the dye, shake the bed for about 15 min.
  12. Observe the result, open the instrument and software, wipe the sample stage with alcohol, put the result on the sample stage, choose blue light to observe the result and save it. Remove the sample, wipe the sample with alcohol and switch off the instrument.

    13. Experiment result:


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Wet Lab Notes Part II
Experiment topic: Slide Preparation, Chamber Adhesion, and Single-Molecule Platform Detection Operations

Experiment Period：2024.4.7

Experimenters: Chu Zibin, She Ao, Wang Ruiying, Huang Lanxin, Cai Leyan

Experiment Purpose:

To prepare materials and equipment for subsequent characterization of the single-molecule platform.

Experimental Procedures:

1. Slide Preparation

  1. Place the slides in a dyeing jar, add 1 mol/L KOH solution to immerse the slides, and ultrasonic for 20 minutes.
  2. Pour out the KOH solution and wash the slides twice with deionized water.
  3. Immerse the slides in chromatography-grade methanol and ultrasonic for 15 minutes (wrap with aluminum foil to reduce evaporation), then wash with acetone twice.
  4. Pour out the acetone, prepare a mixture of 49 mL acetone and 1 mL APTES, and add it to the dyeing jar to immerse the slides, place in the dark for 10 minutes, ultrasonic for 1 minute, place in the dark again for 10 minutes, pour out the solution, wash the slides twice with deionized water, and dry with nitrogen (tilt slightly to the right, feel the airflow size, adjust to avoid breaking the slides). (The graduated cylinder and beaker used to prepare the solution should be rinsed with acetone in advance.)
  5. Pour deionized water into an empty pipette tip box to immerse the bottom, and place the dried slides on the support inside the tip box.
  6. Cut the tip of the pipette about 0.5 cm, narrow end up, prepare AB glue (mix AB glue in a 1:1 ratio), use tweezers to coat the outer wide end with glue and attach it to the slide, and drop the sample after the glue solidifies.

2. Single-Molecule Microscope (Use in Complete Dark Room)

  1. Hardware: Turn on the 4 switches from right to left and top to bottom; Software: Open the yellow, black, and three consecutive software below, and use it when the temperature reaches -80°C.
  2. Drop oil onto the lens, place the slide on and align it with the lens, turn on the red light and light shield, adjust the wide field, and use the coarse focus to make the image clearer, appear bright spots, then adjust the TIRF angle to make the image clear, take pictures and save, select 3 clear fields for dynamic capture of 100 images each sample.
  3. Use the self-developed fluorescence point counting program to analyze the captured images.
  4. Close the small window first and select ”No,” then close the large window and select ”No,” in the reverse order of opening. Wipe the lens with lens paper, then use a cotton swab to gently clean, restore the lens, reset the light shield, and cover it.
Notes
  1. When drying the slides with nitrogen, be mindful of the airflow size to avoid breaking the slides.
  2. The chamber must be pressed tightly with tweezers while the glue is not dry to ensure there are no gaps.
  3. The chamber must be pressed tightly with tweezers while the glue is not dry to ensure there are no gaps.
  4. Clean the optical components to prevent dust or dirt from affecting image quality.