20q12/20q13.1

Genebiosolution > FishBio Probes > 20q12/20q13.1

Product Cat. No.: GBS-020

For Research Use Only.

20q deletion probe reagent Instruction Manual

Product Name

20q deletion probe reagent.

Package Specifications

10 Tests/box

Product Introduction

The kit uses orange fluorescein-labeled 20q12 probe and green fluorescein-labeled 20q13.1 probe to bind 20q12/20q13.1 probe to the target detection site by in situ hybridization.

Product Composition

The kit consists of 20q12/20q13.1 dual color probe as shown in Table 1.

Table 1 Kit composition

Component name Specifications Quantity Main components
20q12/20q13.1 dual color probe 100μL/Tube 1 20q12 Orange probe 20q13.1 Green probe

Storage conditions & Validity

Keep sealed away from light at -20°C ±5°C. The product is valid for 12 months. Avoid unnecessary repeated freezing and thawing, which should not exceed 10 times. After opening, for short-term preservation within 24 hours, keep sealed at 2-8°C in the dark. For long-term preservation after opening, keep the lid sealed at -20°C ±5°C away from light. The kit is transported below 0°C.

Applicable Instruments

Fluorescence microscopy imaging system including fluorescence microscopy and filter sets suitable for DAPI (367/452), Green (495/517), and Orange (547/565).

Sample Requirements

  1. Applicable specimen types: Fresh bone marrow samples that have not been fixed are stored at 4°C for less than 24 hours; bone marrow cell suspension after fixation is stored at -20°C for less than 6 months; the prepared bone marrow cell slides can be set at -20°C for less than 1 month.
  2. When specimens are stored at too high or too low a temperature (e.g., frozen), the specimen will not be used for testing and should be discarded.
  3. If the cell suspension is excessively volatile or contaminated during storage, the sample should be discarded.

Related Reagents

The following reagents are required for the experiment but not provided in this kit

  1. 20×SSC, pH 5.3±0.2
    Weigh 176g of sodium chloride and 88g of sodium citrate, dissolve in 800mL of deionized water, adjust the pH to 5.3±0.2 at room temperature, and complete to 1L with deionized water. High-pressure steam sterilization, stored at 2-8°C. The solution shelf life is 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
  2. 2×SSC, pH 7.0±0.2
    Take 100mL of the above 20×SSC, dilute with 800mL deionized water, mix, adjust the pH to 7.0±0.2 at room temperature, complete to 1L with deionized water, and store at 2-8°C. The shelf life is 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
  3. Ethanol Solution: 70% ethanol, 85% ethanol
    Dilute 700mL and 850mL of ethanol with deionized water to 1L. The shelf life is 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
  4. 0.3% NP-40/0.4×SSC solution, pH 7.0-7.5
    Take 0.6mL NP-40 and 4mL 20×SSC, add 150mL deionized water, mix, adjust the pH to 7.0-7.5 at room temperature, and complete to a volume of 200mL with deionized water. Store at 2-8°C. The shelf life is 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
  5. Fixation solution (methanol: glacial acetic acid = 3:1)
    Prepare a ready-to-use fixation solution by mixing 30mL of methanol and 10mL of glacial acetic acid thoroughly.
  6. 0.075M KCl solution
    Weigh 2.8g of potassium chloride, dissolve in 400mL of deionized water, and complete to 500mL with deionized water. Store at room temperature. The solution shelf life is 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
  7. Diamidinyl phenylindole (DAPI) counterstain
    Use a commercially available anti-quenching DAPI counterstain.

Instructions

  1. Sample collection and slides preparation
    1. Sample collection: Take 3mL of anticoagulated bone marrow cell samples.
    2. Cell harvesting: Place 3mL of anticoagulated peripheral blood in a 15mL centrifuge tube, centrifuge at 500g for 5 min, carefully discard the supernatant, and resuspend about 500μL of the residue.
    3. Cell washing: Add 5mL of 1×PBS buffer, mix and resuspend the cell pellet, centrifuge at 500g for 5 min, carefully discard the supernatant, and resuspend the cells with about 500μL of the residue; repeat 1 time.
    4. Cells hypotonicity: Add 10mL of hypotonic solution pre-warmed to 37°C and place in a water bath at 37°C for 15-20 min.
    5. Cells pre-fixation: Pre-fix the cells by adding 1mL (10% by volume) of fixative solution to the cell suspension after the completion of hypotonic osmosis. Gently pipette, mix and centrifuge for 5 min at 500g, discard the supernatant, and resuspend about 500μL of the residue.
    6. Cell fixation: Slowly add 10mL of fixative solution to the cell suspension at room temperature for 10 min, centrifuge at 500g for 5 min, and resuspend the cells with about 500μL of the residue; repeat once (the cells may be fixed several times until the cell pellet is washed and cleaned).
    7. Cell suspension preparation: Pipet the supernatant and add the appropriate amount of fixative solution to prepare the appropriate cell suspension concentration.
    8. Slides preparation: Pipet 3-5μL of cell suspension onto the slides and place at 56°C for 30 min.
  2. Slides pretreatment
    1. At room temperature, rinse the glass slides twice with 2×SSC (pH 7.0) solution for 5 min each time.
    2. Place the glass slides in 70% ethanol, 85% ethanol, and 100% ethanol and dry for 2 minutes.
  3. Denaturation and Hybridization
    The following operations should be performed in a darkroom.

    1. Take out the probe put at room temperature for 5min. Mix and centrifuge briefly. Take 10μl droplet in the cell and drop in the hybridization zone,immediately cover 22mmx22mm glass slide area; spread evenly without bubbles the probe under the glass slide covered area and seal edges with rubber (edge sealing must be thorough to prevent dry film from affecting the test results during hybridization).
    2. Place the glass slides in the hybridization instrument, denature at 88°C for 2 minutes (the hybridizer should be preheated to 88°C), and hybridize at 45°C for 2 to 16 hours.
  4. Washing
    The following operations should be performed in a darkroom.

    1. Take out the hybridized glass slides, remove the rubber on the coverslip, and immediately immerse the slides in a 2×SSC solution for 5 seconds to remove the coverslip.
    2. Place the slides in 2×SSC at room temperature for 1 min.
    3. Take out the slides and immerse them in a preheated (68°C) 0.3% NP-40/0.4×SSC solution and wash for 2 min.Preparation of 0.3% NP-40/0.4×SSC: For 1L preparation, take 3mL NP-40 and 20mL 20×SSC, dissolve fully, mix well, and use 1M NaOH to adjust the pH to 7.2.
    4. Remove the slides and immerse them in preheated (37°C) deionized water, wash for 1 min, and dry the slides naturally in the dark.
  5. Counterstaining
    The following operations should be performed in a darkroom
    10μL DAPI compound dye is dropped in the hybridization area of the glass slide and immediately covered. The suitable filter is selected for glass slide observation under the fluorescence microscope.
  6. FISH results observation
    Place the counterstained film under the fluorescence microscope, and first put it under the low-power objective lens (10x) confirm the cell area under the microscope; Go to 40x under the objective lens, find a position where the cells are evenly distributed; Then in the high-power objective (100x) the FISH results of nuclei were observed.

Precautions

  1. The results of this kit may be affected by various factors, including the sample itself, hybridization temperature and time, operating environment, and the limitations of current molecular biology technology, which may lead to incorrect results.
  2. Users must understand the potential errors and accuracy limitations that may exist in the detection process.
  3. All chemicals are potentially hazardous. Avoid direct contact. Used kits are considered clinical waste and should be properly disposed of.
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