用于生产用于DNA测序的M13噬菌体和单链DNA的改进方法。

Improved method for the production of M13 phage and single-stranded DNA for DNA sequencing.

作者信息

Reddy P, McKenney K

机构信息

Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.

出版信息

Biotechniques. 1996 May;20(5):854-6, 858, 860. doi: 10.2144/96205st05.

DOI:10.2144/96205st05

PMID:8723931

Abstract

An improved method is described for the efficient production of M13 phage and M13 single-stranded (ss)DNA in a relatively short time period. Infection of E. coli (F') cells with as few as 5 phage particles can yield 10(12) phage particles/mL in 3 hours if the cells are grown in LB broth or SOB broth supplemented with about 5 mM Mg2+. The method tolerates large variations in the initial multiplicity of infection (5-5000 phage per 5 x 10(7) cells) and still yields about 10(12) phage particles/mL. These amounts are sufficient to purify 10-15 micrograms of ssDNA and to carry out at least 10-15 DNA sequencing reactions.

摘要

本文描述了一种改进方法，可在相对较短的时间内高效生产M13噬菌体和M13单链（ss）DNA。如果在补充了约5 mM Mg2+的LB肉汤或SOB肉汤中培养细胞，用少至5个噬菌体颗粒感染大肠杆菌（F'）细胞，3小时内可产生10(12)个噬菌体颗粒/毫升。该方法可耐受初始感染复数的大幅变化（每5×10(7)个细胞5 - 5000个噬菌体），仍可产生约10(12)个噬菌体颗粒/毫升。这些产量足以纯化10 - 15微克的ssDNA，并进行至少10 - 15次DNA测序反应。