A high-throughput, low-cost method for the preparation of "sequencing-ready" phage DNA template.
We have developed a simple and efficient protocol for the isolation of good-quality recombinant phage DNA useful for all downstream processing, including automated sequencing. The overnight-grown phage particles were effectively precipitated (without any contaminating Escherichia coli DNA and other culture media components) by adjusting the pH of the culture medium to 5.2 with sodium acetate, followed by addition of ethanol to 25%. The phage DNA was selectively precipitated with ethanol in the presence of guanidinium thiocyanate under alkaline pH, resulting in uniform quality and quantity of phage DNA. The quality of the phage DNA preparation was demonstrated by DNA sequencing that provided an average read length of >700 bases (PHRED20 quality). This protocol for plating, picking, growing, and subsequent DNA purification of individual phage clones can be completely automated using any standard robotic platform. This protocol does not require any commercial kits and can be completed within 2h.
Reddy
Palakolanu Sudhakar
PS
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Nair
Suresh
S
Mallikarjuna
Garladinne
G
Kaul
Tanushri
T
Markandeya
Gorantla
G
Sopory
Sudhir K
SK
Reddy
Malireddy K
MK
D010582:Bacteriophage lambda / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015723:Gene Library; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic
D004279:DNA, Viral