Green Michael R, Sambrook Joseph
Cold Spring Harb Protoc. 2017 Nov 1;2017(11):pdb.prot093419. doi: 10.1101/pdb.prot093419.
Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected bacteria into the surrounding medium. Several methods are available to purify the polymorphic filamentous particles. In this protocol, the particles are concentrated by precipitation with polyethylene glycol (PEG) in the presence of high salt. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. The resulting preparation is pure enough to be used as a template for DNA sequencing. A yield of 5-10 µg of single-stranded DNA/mL of infected cells may be expected from recombinant bacteriophages bearing inserts of 300-1000 nt.
噬菌体M13单链DNA是从受感染细菌分泌到周围培养基中的病毒颗粒中制备的。有几种方法可用于纯化多态性丝状颗粒。在本实验方案中，通过在高盐存在下用聚乙二醇（PEG）沉淀来浓缩颗粒。随后用苯酚提取释放出单链DNA，然后通过乙醇沉淀收集。所得制剂纯度足以用作DNA测序的模板。带有300 - 1000 nt插入片段的重组噬菌体预期每毫升感染细胞可产生5 - 10 μg单链DNA。
