Experiments

HCR
  1. Add tap water until the drainboard at the bottom of the can is slightly submerged.
  2. Place 1.5 mL tubes in a sterile bag and place it on top of the drainboard.
  3. Select Sterilize and Keep Warm and start at 121 °C and 15 minutes.
  4. Wait approximately 1 hour and 15 minutes.

Ref. Tomy Seiko Co., Ltd. (n.d.). What is Autoclave? [What is an autoclave (high-pressure steam sterilizer)?]. https://bio.tomys.co.jp/topics/detail/01097.php

  1. Prepare agarose gel (1%).
    1. Add 400 mL of 1x TAE to an Erlenmeyer flask.
    2. Add 4 g of agarose.
    3. Heat the solution by microwave until it boils, while stirring appropriately.
    4. After the solution has cooled to 50 ℃, add 16 μL of Midori Green solution.
    5. Pour the solution into the plate and leave for one hour.
    6. Store the gel on a kimtowel moistened with nuclease free water.
  2. Place the prepared gel on the gel bed of the migration tank.
  3. Pour buffer solution to a level of about 3 mm above the surface of the gel.
  4. Apply 10 µL of the sample to the wells.
  5. Start migration.
  6. After preparing the dye in a sealable container, remove the gel tray from the migration tank and submerge it in the stain solution.
  7. After allowing it to stand for the required amount of time, remove the gel and place it on the transilluminator for observation.

Ref. Takara Korea Biomedical Inc. (n.d.). Mupid-2 Plus Instruction Manual. https://www.takara.co.kr/file/manual/pdf/Mupid%202plus%20manual.pdf

  1. Check that the cleaning water bottle contains Dw and that the waste liquid tank is not full, and then clean the probe.
  2. Perform microchip cleaning.
  3. Register the samples on the capillary electrophoresis device MultiNA and set the wells to contain the samples.
  4. Dilute a solution of the fluorescent dye (diluted 100 times with TE) 100 times with DNA-500 (gel) to create a separation buffer.
  5. Place the calculated amount of the DNA marker displayed on MultiNA into a 0.5 mL tube.
  6. Cut the 8-tube strips into 2-tube strips, and add 15 µL of the size ladder to one tube.
  7. Set the cleaning solution, separation buffer, DNA marker, and size ladder in the designated positions.
  8. Dispense 10 µL of the electrophoresis samples into 8-tube strips and place them in the wells set in step 3.
  9. Start analysis.

Ref. Shimadzu Corporation. (n.d.). Microchip Electrophoresis System for DNA/RNA Analysis MCE-202 MultiNA Instruction Manual. http://mor.niboch.nsc.ru/public/MBA_Course/References/Electrophoresis/devices/Shimadzu%20MultiNA%20manual.292-28464D_SWmanualMCE-202(E).pdf

  1. Blank with 1 µL of TE.
  2. Run 1 µL of samples.
  3. Rinse with 1 µL of TE after each sample.
  1. Set up the QuantStudio 3 Real-Time PCR System. Modify experiment settings as needed.
  2. Load a 96well plate in the instrument.
  3. Start the run.

Ref. Thermo Fisher Scientific. (n.d.). QuantStudio 3 & 5 Real-Time PCR Systems. https://assets.fishersci.com/TFS-Assets/LSG/manuals/MAN0010407_QuantStudio3_5_InstallUseMaint_UG.pdf

  1. Prepare HCR Buffer (10 mL) in 15 mL centrifuge tubes:
    2. HCR Buffer Component
  1. Diluted 0.7813 g of KCl with 10.48 mL of Dw to prepare 1 M KCl aq.
  2. Diluted 2.6579 g of MgSO4・7H2O with 9.422 mL of Dw to prepare 1 M MgSO4 aq.
  3. Diluted 1.5798 g of (NH4)2SO4 with 10.0021 mL of Dw to prepare 1 M (NH4)2SO4 aq.
  4. Prepared 10x LT Buffer in 15 mL centrifuge tube:
    5. LT Buffer Component
  1. Prepared 64 mM HEPES-NaOH (pH 7.7).
  2. Diluted 2.5 µL of CoCl2 aq. (4 mM) with 97.5 µL of Nfw to prepare 100 µM solution.
  3. Diluted 10 µL of CoCl2 aq. (100 µM) with 990 µL of Nfw to prepare 1 µM solution.
  4. Prepared 5x UTokyo Buffer in 15 mL centrifuge tubes:
    5. UTokyo Buffer Component