“Colony Sequencing”: Direct Sequencing of Plasmid DNA from Bacterial Colonies
نویسندگان
چکیده
منابع مشابه
Direct colony sequencing of plasmid DNA by dye terminator methods.
Direct colony sequencing of plasmid DNA is a powerful technique. Radioactive or fluorescent dye primer methods are the reported direct colony sequencing methods (2–4,6). Recently, dye terminator sequencing chemistry (7) has been improved by the introduction of a modified enzyme, AmpliTaq DNA polymerase FS (8), as well as a new d-Rhodamine and BigDye terminator (PE Biosystems, Foster City, CA,...
متن کاملDirect neutralization of alkaline-denatured plasmid DNA in sequencing protocol by the sequencing reagent itself.
Denaturation of the double stranded DNA is required before sequencing by the dideoxy chain termination method (1). Good results are routinely obtained with the alkaline-denaturation method, which involves incubation of the DNA in the presence of 0.2 M NaOH for a few minutes at room or higher temperatures (2,3). The alkaline mixture is then neutralized by the addition of sodium acetate (0.3 M), ...
متن کاملDNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.
The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combinati...
متن کاملRapid single-colony whole-genome sequencing of bacterial pathogens
OBJECTIVES As a result of the introduction of rapid benchtop sequencers, the time required to subculture a bacterial pathogen to extract sufficient DNA for library preparation can now exceed the time to sequence said DNA. We have eliminated this rate-limiting step by developing a protocol to generate DNA libraries for whole-genome sequencing directly from single bacterial colonies grown on prim...
متن کاملComparison of six commercial kits to extract bacterial chromosome and plasmid DNA for MiSeq sequencing
We compared commercial kits for extraction of genomic DNA from the Gram-negative bacterium Klebsiella pneumoniae for subsequent Miseq sequencing. Purification of DNA was based on matrix binding (silica or anion exchange resin) or differential precipitation (salting out), respectively. The choice of extraction kit had little effect on sequencing quality and coverage across drastically different ...
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ژورنال
عنوان ژورنال: BioTechniques
سال: 1997
ISSN: 0736-6205,1940-9818
DOI: 10.2144/97223bm08