Question Detail
Does using low-melting-point agarose for gel extraction have a big impact on the efficiency of PCR products from animal pathogens?
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All Answers4
A
AntibodyPro5159
2026-09-17
低熔点琼脂糖凝胶成胶后熔点约65℃，融胶一般放50-55℃水浴，切胶回收时不要让水浴超过60℃。它的凝固点约25-30℃，所以切好的胶块加入溶胶液后要保温并尽快上柱；室温低时离心管会慢慢变稠，可放50℃水浴回温。跑胶时电压别太高，避免发热把胶融软。
蔡
蔡检疫
2026-09-12
低熔点琼脂糖熔点低，胶回收时可在较低温度下融胶，对1.2kb产物和后续克隆测序更友好，通常能减少高温造成的DNA损伤和回收损失。你们一个月做两三次，用量不大，换低熔点带来的单次成本增加可接受。建议先做一次对比：同一管PCR产物分成两份，分别用普通胶和低熔点胶跑，切回收后用同一试剂盒洗脱，测浓度和纯度再决定。
助
助理老司机8969
2026-09-23
样品量一个月两三次，如果后续克隆测序频繁失败、回收得率低，换低熔点值得。可以先算一笔：低熔点琼脂糖贵一点，但做胶回收用到的小胶体积有限，单次增加成本不大。更重要的是回收片段质量好，减少重做PCR和测序返工。普通琼脂糖回收1.2kb其实也能做，关键在切胶薄、紫外照射时间短、用新鲜TAE。
学
学生老师
2026-07-19
不一定马上换低熔点。普通琼脂糖回收1.2kb效率不高，常见原因是切胶时紫外照太久、胶块过大、溶胶不彻底或溶胶后未冷却到室温就过柱。可以先优化这几步：使用蓝光切胶仪或短时紫外，胶块切成小粒，溶胶后确认清澈，必要时加异丙醇。优化后仍不满意，再换低熔点琼脂糖做平行比较，不要只凭一次回收率下结论。
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