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.
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