MidipreppreparationofPlasmidDNA

实验概要

The  PureLink™ HiPure Plasmid DNA Midiprep Kit allows purification of  100–350 μg of high-quality plasmid DNA from 15–25 mL overnight E. coli  cultures in ~2 hours when cloning high copy number plasmids.

主要试剂

Before Starting

Before  beginning, verify that the Resuspension Buffer (R3) contains RNase A,  and no precipitate has formed in the Lysis Buffer (L7).

Materials Needed

Overnight culture of transformed E. coli cells

Isopropanol

70% ethanol

Sterile, microcentrifuge tubes

PureLink™ Nucleic Acid Purification Rack

Tubes or centrifuge bottles for harvesting cells

Centrifuge and rotor appropriate for harvesting cells

Appropriate 15-mL centrifuge tubes capable of

withstanding centrifugation forces >12,000 × g

Centrifuge capable of centrifuging at >12,000 × g at 47°C

Optional: PureLink™ HiPure Precipitator Module

Components Supplied with the Kit

Resuspension Buffer (R3) with RNase A

Lysis Buffer (L7)

Precipitation Buffer (N3)

Equilibration Buffer (EQ1)

Wash Buffer (W8)

Elution Buffer (E4)

TE Buffer (TE)

HiPure Filter Midi Columns

Column Holder

Note:  The protocol for the Midiprep kit of the PureLink™ HiPure Plasmid  Filter Purification Kits is designed for purification of both high and  low copy number plasmids without the need for adjusting buffer volumes.

实验步骤

1.        Equilibrating the Column

The PureLink™ HiPure Filter Midi Columns are packaged with the Filtration Cartridge pre-inserted into the column housing.

To Equilibrate the column:

1)      Use  the Column Holder to support a HiPure Filter Midi Column in the mouth  of a flask, or place the Midi Column on the PureLink™ Nucleic Acid  Purification Rack (see manual supplied with the rack for more details).

2)      Apply 15 mL Equilibration Buffer (EQ1) directly to the filtration cartridge in the Midi Column.

3)      Allow the solution in the HiPure Filter Midi Column to drain by gravity flow.

4)      Prepare the cell lysate (see below) while the HiPure Filter Midi Column is equilibrating.

2.        Preparing Cell Lysate

1)      Harvest  bacterial culture cells by centrifugation. For high copy number  plasmids, harvest 15–25 mL of an overnight LB culture per sample in a  50-mL disposable tube. For low copy number plasmids, harvest 25–100 mL  of an overnight LB culture per sample in a 50-mL disposable tube.

2)      Centrifuge the cells at 4,000 × g for 10 minutes to harvest the cells. Remove all medium.

3)      Add  10 mL Resuspension Buffer (R3) with RNase A to the cell pellet in the  tube and resuspend the cells. Gently shake the tube until cell  suspension is homogeneous.

4)      Add  10 mL Lysis Buffer (L7). Place the cap on the tube and ensure it is  secure. Mix gently by inverting the capped tube until the lysate mixture  is thoroughly homogenous. Do not vortex.

5)      Incubate the lysate at room temperature for 5 minutes. Do not exceed 5 minutes.

6)      Add  10 mL Precipitation Buffer (N3) and mix immediately by inverting the  tube until the mixture is thoroughly homogeneous. Do not vortex.

7)      Proceed to Loading Filter Column and Washing DNA

3.        Loading Filter Column and Washing DNA

1)      Transfer  the precipitated lysate from Step 6 (above), including all the  precipitated material into the equilibrated HiPure Filter Midi Column.  Let the lysate pass through the filter by gravity flow until the flow  stops (10–15 minutes) or becomes very slow (<1 drop per 10 seconds).  Discard the flow-through. Optional: The final DNA yield  may be increased by washing the residual bacterial lysate in the HiPure  Filter Midi column with 10 mL Wash Buffer (W8). Again, let the buffer  flow through the HiPure Filter Midi Column by gravity flow until the  flow stops or dripping becomes very slow.

2)      Immediately  after the HiPure Filter Midi Column has stopped dripping, remove and  discard the inner Filtration Cartridge from the column. Note: Do not reuse the Filtration Cartridge. The cartridge is designed for single use only.

3)      Wash  the Midi column with 20 mL Wash Buffer (W8). Allow the solution in the  column to drain by gravity flow. Discard the flow-through.

4)      Proceed to Eluting DNA (below).

4.        Eluting DNA

1)      Place a sterile 15-mL centrifuge tube (elution tube) under the HiPure Filter Midi column.

2)      Add 5 mL Elution Buffer (E4) to the Midi column to elute the DNA. Allow the solution to drain by gravity flow. Do not force out any remaining solution. The elution tube contains the purified DNA.

3)      Discard the HiPure Filter Midi column.

4)      Proceed to Precipitating DNA

5.        Precipitating DNA with Isopropanol

1)      Add 3.5 mL isopropanol to the elution tube containing the DNA (see Eluting DNA). Mix well.

2)      Incubate the DNA-isopropanol mixture for 2 minutes at room temperature.

3)      Centrifuge the tube at >12,000 × g for 30 minutes at 4°C. Carefully remove and discard the supernatant.

4)      Resuspend the DNA pellet in 3 mL 70% ethanol.

5)      Centrifuge the tube at >12,000 × g for 5 minutes at 4°C. Carefully remove and discard the supernatant.

6)      Air-dry the pellet for ~10 minutes.

7)      Resuspend  the DNA pellet in 200 μL TE Buffer (TE) for high copy number plasmids.  For low copy number plasmids, use 100 μL TE Buffer (TE).

6.        Storing DNA

Store the purified DNA at –20°C, or proceed to the desired downstream application.

注意事项

1.        For  DNA precipitation using the Midiprep Kit, you can use the PureLink™  HiPure Precipitator Module which allows DNA precipitation within 10  minutes without centrifugation, or you can follow the protocol for  Precipitating DNA with Isopropanol (below) to perform traditional DNA  precipitation using centrifugation. Refer to the manual supplied with  the PureLink™ HiPure Precipitator Module for a detailed protocol.

2.        Occasionally,  insoluble particles may be present. These particles do not influence  the quality of the DNA and can be easily removed. To remove insoluble  particles, centrifuge the DNA solution at high speed for 1 minute at  room temperature Transfer the supernatant (DNA sample) into a fresh  tube.

3.        To  avoid repeated freezing and thawing of DNA, store the purified DNA at  4°C for immediate use or aliquot the DNA and store at –20°C for  long-term storage.