Purification of nucleic acids

• US 8,816,064 B2
• Filed: 09/30/2011
• Issued: 08/26/2014
• Est. Priority Date: 09/30/2010
• Status: Active Grant

• Current Assignee: PhyNexus, Inc.
• Original Assignee: PhyNexus, Inc.
• Inventors: Suh, Chris, Hoang, Lee, Gjerde, Douglas T.
• Primary Examiner(s): LEWIS, PATRICK T
• Application Number:
  US13/250,040
• Publication Number:
  US 20120083598A1
• Time in Patent Office: 1,061 Days
• Field of Search: None
• US Class Current: 536/25.42
• CPC Class Codes: C12N 15/1017 by filtration, e.g. using filters, frits, membranes

Abstract

The present invention solves the problem of isolating nucleic acids from cells in the presence of the growth medium. The invention is particularly useful for isolating extrachromosomal replicons such as plasmids. Cells are lysed in the presence of the medium in which they were grown and nucleic acids are isolated using a pipette tip column. A liquid handling robot can be used to isolate nucleic acids from multiple samples simultaneously without the need for human intervention.

Claims

1. A method for isolating plasmid DNA from cells in a liquid culture, comprising:

a. providing a liquid culture comprised of growth medium and cells, wherein the cells contain plasmid DNA;

b. lysing the cells in the liquid culture in the presence of the growth medium to produce a mixture, wherein the cells are lysed with alkali, and wherein the cells in the liquid culture are not centrifuged prior to lysis, and wherein lysozyme is not used for lysis;

c. providing a pipette tip column; and

d. passing at least a portion of the mixture through the pipette tip column, whereby a portion of the plasmid DNA is captured on the pipette tip column.

2. The method of claim 1, wherein the mixture is passed through the pipette tip column by repeated aspirate and expel steps.

3. The method of claim 2, wherein between step (b) and step (d), a chaotropic solution is added to the mixture, and wherein the pipette tip column is further comprised of a silica solid phase.

4. An automated method for isolating plasmid DNA from cells in a liquid culture, comprising:

a. providing a liquid culture comprised of growth medium and cells, wherein the cells contain plasmid DNA;

b. lysing the cells in the liquid culture in the presence of the growth medium to produce a mixture, wherein the cells in the liquid culture are not centrifuged prior to lysis;

c. providing a pipette tip column; and

d. passing at least a portion of the mixture through the pipette tip column, whereby a portion of the plasmid DNA is captured on the pipette tip column,

wherein steps (b) and (d) are performed at pre-determined times.

5. The method of claim 4, wherein the mixture is passed through the pipette tip column by repeated aspirate and expel steps.

6. The method of claim 5, wherein between step (b) and step (d), a chaotropic solution is added to the mixture, and wherein the pipette tip column is further comprised of a silica solid phase.

7. The method of claim 4, wherein steps (a) and (b) are performed in a microplate.