📚 Key Points in Molecular Biology Laboratory Techniques | 分子生物学实验操作要点
Molecular biology experiments form an essential component of A-Level Biology courses. Mastering the practical skills involved — from pipetting microlitre volumes to running a PCR reaction — not only secures marks in the practical endorsement but also builds a deeper understanding of the underlying theory.
分子生物学实验是 A-Level 生物课程的重要组成部分。掌握相关实验技能——从微升量级的移液操作到运行 PCR 反应——不仅能在实验能力评估中赢得分数，还能加深对背后理论的理解。
1. Laboratory Safety and Preparation | 实验室安全与准备
Before beginning any molecular biology practical, carry out a risk assessment. Identify hazards such as ethidium bromide (a mutagen), UV radiation from transilluminators, and electric shock from electrophoresis equipment. Wear gloves, a lab coat, and safety goggles at all times.
在开始任何分子生物学实验前，务必进行风险评估。识别潜在危害，如溴化乙锭（一种诱变剂）、透照仪发出的紫外辐射以及电泳设备的电击风险。始终佩戴手套、实验服和护目镜。
Prepare all materials in advance. Label every tube with the sample name, concentration, date, and your initials. Check that water baths and thermal cyclers are at the correct temperature before use.
提前准备所有材料。在每个离心管上标注样品名称、浓度、日期和姓名缩写。使用前检查水浴锅和 PCR 仪的温度是否准确。
2. Micropipetting Mastery | 移液器操作要点
The micropipette is the most used tool in molecular biology. Select the correct size: a P1000 measures 200–1000 μL, a P200 measures 20–200 μL, and a P10 measures 0.5–10 μL. Never use a pipette outside its stated range.
移液器是分子生物学中使用频率最高的工具。选择合适量程：P1000 量程为 200–1000 μL，P200 为 20–200 μL，P10 为 0.5–10 μL。切勿在标称量程之外使用移液器。
When aspirating, depress the plunger to the first stop, immerse the tip just below the liquid surface, and release slowly. To dispense, press to the first stop, pause, then press to the second stop to expel residual liquid. Hold the pipette at a 10–45° angle and avoid touching the container walls.
吸液时，将活塞按至第一档，枪头刚好浸入液面下方，缓慢松开。排液时，按至第一档，停顿片刻，再按至第二档以排出残留液体。移液器应保持 10–45° 倾角，避免触碰容器壁。
Always use a fresh tip for each sample to prevent cross-contamination. Pre-wet the tip by aspirating and dispensing the same liquid once before the actual measurement — this increases accuracy for viscous solutions.
每个样品必须更换新枪头，以防交叉污染。在正式测量前，可先吸排一次同种液体以预润湿枪头——这能提高粘稠溶液的准确性。
3. DNA Extraction and Purification | DNA 提取与纯化
DNA extraction typically follows the sequence: cell lysis, removal of proteins and lipids, and precipitation of DNA. For plant tissue such as strawberries or kiwi, mash the tissue in a buffer containing detergent (to disrupt cell membranes) and salt (to help DNA precipitate).
DNA 提取通常遵循以下流程：细胞裂解、去除蛋白质和脂质、沉淀 DNA。以草莓或猕猴桃等植物组织为例，将组织在含去污剂（破坏细胞膜）和盐（帮助 DNA 沉淀）的缓冲液中研磨。
After lysis, filter the mixture to remove debris. Add ice-cold ethanol or isopropanol; DNA is insoluble in alcohol and will precipitate at the interface. Spool the DNA onto a glass rod or collect it by centrifugation at high speed.
裂解后，过滤混合物以去除残渣。加入冰乙醇或异丙醇；DNA 不溶于醇类，会在界面处沉淀。可用玻璃棒缠绕挑出 DNA，或通过高速离心收集。
For higher purity (required for PCR or restriction digestion), use a commercial spin-column kit. These columns use a silica membrane that binds DNA in high-salt conditions and releases it in low-salt buffer or water.
如需更高纯度（用于 PCR 或限制性酶切），可使用商品化离心柱试剂盒。这类离心柱利用硅胶膜：在高盐条件下结合 DNA，在低盐缓冲液或水中释放 DNA。
Assess DNA quality by measuring the A₂₆₀/A₂₈₀ ratio. A ratio of 1.8 indicates pure DNA; a lower ratio suggests protein contamination, while a higher ratio may indicate RNA contamination.
通过测定 A₂₆₀/A₂₈₀ 比值评估 DNA 质量。比值为 1.8 表示 DNA 较纯；比值偏低提示蛋白质污染，偏高则可能含有 RNA 污染。
4. Nucleic Acid Quantification | 核酸定量
Use a spectrophotometer or a NanoDrop to measure the absorbance of a DNA sample at 260 nm. An absorbance of 1.0 at A₂₆₀ corresponds to approximately 50 μg/mL of double-stranded DNA, 40 μg/mL of RNA, and 33 μg/mL of single-stranded DNA.
使用分光光度计或 NanoDrop 测量 DNA 样品在 260 nm 处的吸光度。A₂₆₀ 为 1.0 时，双链 DNA 约为 50 μg/mL，RNA 约为 40 μg/mL，单链 DNA 约为 33 μg/mL。
The concentration is calculated using the Beer-Lambert law:
c = (A₂₆₀ × dilution factor × 50) μg/mL
浓度可通过比尔-朗伯定律计算：
c = (A₂₆₀ × 稀释倍数 × 50) μg/mL
When using a NanoDrop, only 1–2 μL of sample is needed. Record the A₂₆₀/A₂₈
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