Method and device for sample preparation

• US 8,377,715 B2
• Filed: 04/26/2010
• Issued: 02/19/2013
• Est. Priority Date: 07/14/2003
• Status: Expired due to Term

• Current Assignee: PhyNexus, Inc.
• Original Assignee: PhyNexus, Inc.
• Inventors: Suh, Chris, Hoang, Lee, Grant, Jennifer E., Gjerde, Douglas T.
• Primary Examiner(s): Menon, Krishnan S
• Assistant Examiner(s): Gerido, Dwan A
• Application Number:
  US12/767,659
• Publication Number:
  US 20100200509A1
• Time in Patent Office: 1,030 Days
• Field of Search: 422/502, 422/524, 422/535, 422/534, 422/527, 436/161, 436/177, 436/178
• US Class Current: 436/177
• CPC Class Codes: B01D 15/3804 Affinity chromatography
• CPC Class Codes: B01J 20/285 based on polymers
• CPC Class Codes: B01J 20/286 Phases chemically bonded to a substrate, e.g. to silica or to polymers
• CPC Class Codes: B01J 20/291 Gel sorbents
• CPC Class Codes: B01J 20/3244 Non-macromolecular compounds
• CPC Class Codes: B01J 2220/54 Sorbents specially adapted for analytical or investigative chromatography
• CPC Class Codes: B01J 2220/58 Use in a single column
• CPC Class Codes: B01J 2220/64 In a syringe, pipette, e.g. tip or in a tube, e.g. test-tube or u-shape tube
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 2035/1053 for separating part of the liquid, e.g. filters, extraction phase
• CPC Class Codes: G01N 30/02 Column chromatography
• CPC Class Codes: G01N 30/603 retaining the stationary phase, e.g. Frits
• CPC Class Codes: G01N 30/6065 with varying cross section
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

The invention provides pipette tip extraction columns for the purification of a DNA vector from un-clarified cell lysate containing cell debris as well as methods for making and using such columns. The columns typically include a bed of extraction media positioned in the pipette tip column, above a bottom frit and with an optional top frit.

Claims

1. A method of extracting plasmid DNA from an unclarified lysate, comprising the steps of:

a. providing an unclarified cell lysate;

b. providing at least one pipette tip column, wherein the pipette tip column is comprised of

i) a column body having an open upper end, an open lower end, and an open channel between the upper and lower ends of the column body, wherein the column body is comprised of a modified pipette tip;

ii) a bottom frit extending across the open channel, said bottom frit having a thickness in the range of 20 μ

m to 1000 μ

m and a pore size in the range of 50 μ

m to 250 μ

m; and

iii) a bed of extraction media positioned inside the open channel and in contact with the bottom frit;

c. passing the unclarified cell lysate through the pipette tip column wherein the plasmid DNA is captured by the bed of extraction media;

d. passing wash solution through the pipette tip column; and

e. eluting the plasmid DNA by passing a desorption solvent through the pipette tip column.

2. The method of claim 1, wherein the upper end of the column is operatively attached to a pump, and wherein the unclarified cell lysate, wash solution and desorption solution are passed through the column by aspirating and expelling through the open lower end of the column.

3. The method of claim 2, wherein the unclarified cell lysate, wash solution or desorption solution are passed back and forth through the bed of extraction media more than once.

4. The method of claim 3, wherein the method is automated and wherein the method is performed in parallel on a plurality of pipette tip columns.

5. A method of extracting plasmid DNA from an unclarified lysate, comprising the steps of:

a. providing an unclarified cell lysate;

b. providing at least one pipette tip column, wherein the pipette tip column is in operative engagement with a pipette or liquid handler, and wherein the pipette tip column is comprised of

i) a column body having an open upper end, an open lower end, and an open channel between the upper and lower ends of the column body, wherein the column body is comprised of a modified pipette tip;

ii) a bottom frit extending across the open channel, said bottom frit having a pore size sufficiently large to allow cell debris to flow through the frit; and

iii) a bed of extraction media positioned inside the open channel and in contact with the bottom frit;

c. passing the unclarified cell lysate back and forth repeatedly through the pipette tip column;

d. passing wash solution through the pipette tip column; and

e. eluting the plasmid DNA by passing a desorption solvent through the pipette tip column.

6. The method of claim 5, wherein the wash solution or the desorption solution are passed back and forth through the bed of extraction media more than once.

7. The method of claim 6, wherein the method is automated and wherein the method is performed in parallel on a plurality of pipette tip columns.