Filtration processes, kits and devices for isolating plasmids
First Claim
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1. A process for isolating plasmid DNA from plasmid-containing cells, comprising the steps of:
- a) washing plasmid-containing cells by filtration with a wash solution to disrupt the cell membrane and degrade RNA;
b) incubating the resulting washed plasmid-containing cells with a lysis/denaturation solution to lyse the cells and denature DNA;
c) incubating the product of step b) with a renaturation solution for an appropriate period of time and at an appropriate temperature to yield a renatured mixture containing dissolved plasmid DNA, insoluble linear DNA and cellular debris;
d) filtering the product of step c) to obtain a filtrate from which insoluble linear DNA and cellular debris have been removed; and
e) obtaining plasmid DNA from the filtrate, wherein no centrifugation step is used in the process.
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Abstract
Processes, kits and preferred devices for rapidly isolating large numbers of plasmid DNAs from plasmid containing cells and for performing high throughput DNA sequencing are described.
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Citations
37 Claims
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1. A process for isolating plasmid DNA from plasmid-containing cells, comprising the steps of:
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a) washing plasmid-containing cells by filtration with a wash solution to disrupt the cell membrane and degrade RNA; b) incubating the resulting washed plasmid-containing cells with a lysis/denaturation solution to lyse the cells and denature DNA; c) incubating the product of step b) with a renaturation solution for an appropriate period of time and at an appropriate temperature to yield a renatured mixture containing dissolved plasmid DNA, insoluble linear DNA and cellular debris; d) filtering the product of step c) to obtain a filtrate from which insoluble linear DNA and cellular debris have been removed; and e) obtaining plasmid DNA from the filtrate, wherein no centrifugation step is used in the process. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A process for isolating plasmid DNA from plasmid-containing cells, comprising the steps of:
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a) washing plasmid-containing cells by filtration with a wash solution to disrupt the cell membrane and degrade RNA; b) incubating the resulting washed plasmid-containing cells with a lysis/denaturation solution to lyse the cells and denature DNA; c) incubating the product of step b) with a renaturation solution for an appropriate period of time and at an appropriate temperature to yield a renatured mixture containing dissolved plasmid DNA, insoluble linear DNA and cellular debris; d) filtering the product of step c) to obtain a filtrate from which insoluble linear DNA and cellular debris have been removed; and e) obtaining plasmid DNA from the filtrate, wherein; the process is performed in a multi-well filtration device; and prior to step a), plasmid containing cells are grown in wells of a multi-well filter plate. - View Dependent Claims (13, 14, 15)
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16. A high throughput DNA sequencing method, comprising the steps of:
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i) growing plasmid containing cells wherein the plasmids comprise DNA to be sequenced; ii) washing plasmid-containing cells by filtration with a wash solution to disrupt the cell membrane and degrade RNA; iii) incubating the resulting washed plasmid containing cells with a lysis/denaturation solution to lyse the cells and denature DNA; iv) incubating the product of step c) with a renaturation solution for an appropriate period of time and at an appropriate temperature to yield a renatured mixture containing dissolved plasmid DNA, insoluble linear DNA and cellular debris; v) filtering the product of step d) to obtain a filtrate from which insoluble linear DNA and cellular debris have been removed; vi) obtaining plasmid DNA from the filtrate; and vii) analyzing a nucleic acid fragment obtained from the plasmid DNA by mass spectrometry to determine the sequence of the fragment of DNA of the plasmid. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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37. A high throughput method, comprising the steps of:
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i) growing plasmid containing cells wherein the plasmids comprise DNA to be sequenced; ii) washing plasmid-containing cells by filtration with a wash solution to disrupt the cell membrane and degrade RNA; iii) incubating the resulting washed plasmid containing cells with a lysis/denaturation solution to lyse the cells and denature DNA; iv) incubating the product of step c) with a renaturation solution for an appropriate period of time and at an appropriate temperature to yield a renatured mixture containing dissolved plasmid DNA, insoluble linear DNA and cellular debris; v) filtering the product of step d) to obtain a filtrate from which insoluble linear DNA and cellular debris have been removed; vi) obtaining plasmid DNA from the filtrate; and vii) sequencing the plasmid DNA or a nucleic acid fragment obtained from the plasmid DNA, whereby a plurality of plasmid samples are sequenced.
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Specification