Methods and devices for nucleic acid purification

• US 8,921,539 B2
• Filed: 03/29/2012
• Issued: 12/30/2014
• Est. Priority Date: 03/29/2011
• Status: Active Grant

• Current Assignee: PhyNexus, Inc.
• Original Assignee: PhyNexus, Inc.
• Inventors: Suh, Chris, Hoang, Lee, Gjerde, Douglas T.
• Primary Examiner(s): MARSCHEL, ARDIN H
• Application Number:
  US13/434,656
• Publication Number:
  US 20120252115A1
• Time in Patent Office: 1,006 Days
• Field of Search: 536/25.4, 435/283.1
• US Class Current: 536/25.4
• CPC Class Codes: B01D 15/322 Normal bonded phase
• CPC Class Codes: B01D 15/361 Ion-exchange
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 3/0275 Interchangeable or disposable dispensing tips
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: G01N 2035/1039 Micropipettes, e.g. microcapillary tubes

Abstract

The invention provides pipette tip columns and automated methods for the purification of nucleic acids such as plasmids from unclarified cell lysates containing cell debris as well as methods for making and using such columns. The columns typically include a bed of medium positioned in the pipette tip column, above a bottom frit and with an optional top frit.

Claims

1. An automated method for capturing plasmid DNA from an unclarified lysate with a pipette tip column, the method comprising:

a) providing a multi-well plate, wherein a plurality of wells in the multi-well plate contain unclarified lysates having a sample volume, wherein each unclarified lysate is comprised of plasmid DNA, liquid and particulates;

b) providing a plurality of pipette tip columns arranged in a 9 mm center-to-center format wherein each pipette tip column is comprise of

i) a column body having an open upper end,

ii) an open lower end,

iii) an open channel between the open upper end and the open lower end,

iv) a bottom frit extending across the open lower end, and

v) a solid phase positioned inside the open channel and above the bottom frit;

c) engaging the open upper end of the pipette tip columns with a pump, wherein the pump is a syringe pump or the head of a robotic liquid handler;

d) submerging the open lower end of the pipette tip columns in the unclarified lysates;

e) aspirating and expelling a portion of the unclarified lysates through the open lower end of the pipette tip columns, wherein the portion of each unclarified lysate aspirated is between 10% and 80% of the sample volume;

f) aspirating and expelling a wash solution through the open lower end of the pipette tip columns;

g) transferring the pipette tip columns into a custom 9 mm center-to-center block, wherein the block is comprised of channels and wherein each channel is comprised of a seal, wherein the channels completely surround each individual pipette tip column over substantially the length of the column, and whereby each individual pipette tip column is sealed within a channel; and

h) applying software-controlled vacuum through the block, wherein the vacuum is applied evenly through each pipette tip column.

2. The method of claim 1, wherein the portion of the unclarified lysate is between 10% and 60% of the sample volume.

3. The method of claim 1, wherein the method is performed on at least 2 and at most 96 pipette tip columns simultaneously.

4. An automated method for capturing plasmid DNA from an unclarified lysate with a pipette tip column, the method comprising:

a) providing a multi-well plate, wherein a plurality of wells in the multi-well plate contain unclarified lysates having a sample volume, wherein each unclarified lysate is comprised of plasmid DNA, liquid and particulates;

b) providing a plurality of pipette tip columns arranged in a 9 mm center-to-center format wherein each pipette tip column is comprised of

i) a column body having an open upper end,

ii) an open lower end,

iii) an open channel between the open upper end and the open lower end,

iv) a bottom frit extending across the open lower end, and

v) a solid phase positioned inside the open channel and above the bottom frit;

c) engaging the open upper end of the pipette tip columns with a pump, wherein the pump is a syringe pump or the head of a robotic liquid handler;

d) submerging the open lower end of the pipette tip columns in the unclarified lysates;

e) aspirating and expelling a portion of the unclarified lysates through the open lower end of the pipette tip columns, wherein the portion of each unclarified lysate aspirated is between 10% and 80% of the sample volume;

f) aspirating and expelling a wash solution through the open lower end of the pipette tip columns;

g) transferring the pipette tip columns into a custom 9 mm center-to-center block, wherein the block is comprised of channels, wherein the channels are comprised of a seal, whereby the channels completely surround each individual pipette tip column and whereby each column is sealed within a channel, and wherein the block is designed to apply vacuum evenly through all the pipette tip columns; and

j) applying software-controlled vacuum through the block, wherein the vacuum is applied evenly through each pipette tip column.

5. The method of claim 4, wherein the portion of the unclarified lysate is between 10% and 60% of the sample volume.

6. The method of claim 4, wherein the method is performed on at least 2 and at most 12 pipette tip columns simultaneously.