Daum H A, White H W, Seidell C M, Johnson P A
FMC Bio-Products, Rockland, ME 04841.
Biotechniques. 1991 Dec;11(6):784-6, 788, 790-1.
Large DNA fragments (greater than or equal to 1 kb), separated in low melting temperature SeaPlaque GTG agarose gels, can be enzymatically processed directly in the presence of this agarose (in-gel). Time saving protocols are discussed for in-gel processing of large DNA fragments in the presence of remelted SeaPlaque GTG agarose, including cloning into pUC18, nick translation, random priming and restriction digestion. These in-gel molecular biology techniques are as efficient as those using DNA recovered from agarose. The effects of UV irradiation, Mg2+ concentration and agarose concentration on selected in-gel protocols are also discussed.
在低熔点温度的SeaPlaque GTG琼脂糖凝胶中分离得到的大于或等于1 kb的大DNA片段，可在这种琼脂糖存在的情况下直接进行酶处理（凝胶内处理）。本文讨论了在重熔的SeaPlaque GTG琼脂糖存在下对大DNA片段进行凝胶内处理的省时方案，包括克隆到pUC18载体中、缺口平移、随机引物延伸和限制性酶切。这些凝胶内分子生物学技术与使用从琼脂糖中回收的DNA的技术一样高效。还讨论了紫外线照射、Mg2+浓度和琼脂糖浓度对所选凝胶内方案的影响。
