1. Core application: Rapid preparation of highly reproducible native PAGE gels, eliminating the tedious calculations and step-by-step gel‑casting procedures of conventional methods.

2. Separation range: Suitable for high-resolution electrophoretic analysis and purification of double-stranded nucleic acids ranging from 10 to 2,000 bp, or single-stranded nucleic acids ranging from 10 to 2,000 nt.

3. Applicable experiments: fine separation of small nucleic acid fragments, SSR molecular marker analysis, primer purity assessment, and nucleic acid purification and recovery, among other experimental applications.

The kit comprises three core functional components, each with distinct storage requirements; therefore, they must be stored separately.

1. 40% Acr-Bis (37.5:1) gel solution: 200 mL per bottle; store at 4°C, protected from light.

2. Gel polymerization catalyst (powder): 3 vials, each containing 0.12 g; store at room temperature before dilution; after dissolution, aliquot and store at −20 °C.

3. TEMED accelerator: 320 μL per vial; store sealed at 4°C.

4. Shipping Instructions: The product is shipped with ice packs to maintain the required temperature. Upon receipt, please open the package and inspect the contents immediately, then store the product according to the specified temperature requirements.

1. Dissolution method: Add deionized water to the centrifuge tube containing the gel polymerization catalyst up to the 1.2 mL mark, gently invert the tube until the powder is completely dissolved, and then mix thoroughly.

2. Storage: After dissolution, aliquot according to single-use amounts and store at −20°C.

3. Contraindications: Repeated freeze–thaw cycles are strictly prohibited, as they can reduce catalyst activity, leading to gelation polymerization failure or uneven polymerization.

Taking the preparation of 10 mL of non-denaturing PAGE gel at different concentrations as an example, the fixed and variable components are added in the following amounts:

Fixed addition amounts (common to all concentrations): 72 μL of dissolved gel polymerization catalyst, 6.4 μL of TEMED, and 1 mL of 10× TBE.

1. 8% gel: 7.42 mL deionized water + 2 mL of 40% Acr-Bis gel‑casting solution;

2. 10% gel: 6.42 mL deionized water + 2.5 mL of 40% Acr-Bis gel‑forming solution;

3. 12% gel: 5.92 mL deionized water + 3 mL of 40% Acr-Bis gel‑forming solution;

4. 15% gel: 5.17 mL deionized water + 3.75 mL of 40% Acr-Bis gel‑forming solution;

5. 20% gel: 3.92 mL deionized water + 5 mL of 40% Acr‑Bis gel‑casting solution.

1. Standard operating procedure: In a clean container, sequentially add deionized water, 10× TBE buffer, 40% acrylamide (37.5:1), the dissolved gel polymerization catalyst, and TEMED. Gently pipette to mix thoroughly, then immediately pour the gel.

2. Reason for adding them last: The gel polymerization catalyst acts as the initiator, and TEMED serves as the accelerator; when added together, they rapidly initiate the polymerization reaction. If added prematurely, the gel will begin to polymerize during the pouring process, resulting in failed casting and an uneven gel structure.

3. Key operational steps: After adding the initiator, mix quickly and gently, taking care to minimize bubble formation; immediately inject the mixture into the glass‑plate sandwich once mixing is complete.

1. Standard polymerization time: Allow to stand at room temperature for approximately 30 minutes, until the gel has fully polymerized and the interface is clear with no signs of fluidity; then it is ready for use.

2. Recommended immediate use: Perform electrophoresis as soon as possible after gelation for optimal separation and resolution.

3. Short-term storage method: If temporary storage is required, wrap the comb in moist cling film and place it in a humid environment at 4°C; do not store for more than 24 hours.

4. Storage precautions: Do not leave the gel exposed for extended periods to prevent dehydration and shrinkage, as well as distortion of the gel pores, which can compromise the quality of electrophoresis bands.

1. Recommended buffer: Electrophoresis must be performed using 1× TBE buffer, which is fully compatible with the gel‑buffer system and ensures stable pH and optimal resolution.

2. TAE is not recommended as a substitute: TAE has low buffering capacity and a different ionic composition from TBE; mixing the two can cause band smearing and abnormal migration rates, severely compromising separation efficiency.

3. Buffer usage: Electrophoresis buffers are not recommended for repeated use; after multiple cycles, the ionic strength decreases and the pH shifts, which can reduce electrophoretic resolution and cause band smearing.

The primary reason for gel non‑polymerization is the failure or insufficient activity of the initiator system; troubleshooting can be conducted in the following sequence:

1. Reagent inactivation: The gel polymerization catalyst or TEMED may have lost its activity. Replace with freshly thawed catalyst and fresh TEMED, and verify that the reagents have not expired or deteriorated.

2. Insufficient addition: The amounts of catalyst or TEMED were not measured accurately. Pipette precisely according to the ratios specified in the manual to avoid measurement errors.

3. Low temperature: Extremely low ambient temperatures can significantly slow down the polymerization rate. You may appropriately raise the operating room temperature, or place the mixture in a 25°C environment to promote polymerization.

The two types of faults correspond to different operational issues and can be diagnosed accordingly:

1. Strip distortion/irregularity First, gel polymerization is uneven because the reagents were not thoroughly mixed before pouring. Second, pulling the comb too forcefully can damage the gel wells; the comb should be removed gently and vertically to avoid tearing the edges of the sample-loading wells.

2. Burning the gel during electrophoresis : Residual acrylamide solution spilled at the bottom of the glass plate can dry and become conductive, leading to a short circuit; after pouring the gel, promptly wipe away any gel solution that has overflowed onto the outer surface of the glass plate with filter paper to prevent residue.

1. Toxicity Warning: The 40% Acr‑Bis gel‑forming solution contains unpolymerized acrylamide monomer, a potent neurotoxin that can be absorbed through the skin and mucous membranes and poses long-term health risks.

2. Personal Protective Equipment: Always wear disposable gloves and a mask when handling this substance, and work in a fume hood or a well-ventilated area to avoid direct contact with skin and eyes.

3. Waste Liquid Disposal: Experimental waste liquids and discarded gels shall be collected and disposed of in accordance with hazardous chemical regulations; unauthorized dumping is strictly prohibited.

4. Post‑polymerization notes: After gel polymerization, the acrylamide monomers crosslink and solidify, significantly reducing toxicity; however, proper handling remains essential to prevent prolonged direct contact.