实验概要
The PureLink™ HiPure Plasmid DNA Maxiprep Kit allows purification of 500–850 μg of high-quality plasmid DNA from 100–200 mL overnight E. coli cultures in ~2 hours when cloning high copy number plasmids.
实验原理
The PureLink™ HiPure Plasmid Filter Purification Kits allow isolation of high yields of highly pure plasmid DNA. The HiPure Filter Column provides rapid clearing of the bacterial lysate without the need for centrifugation. The lysate Filtration Cartridge is integrated into the DNA Binding Column and combines the steps of clearing the bacterial lysate with binding the DNA directly to the anionexchange resin. The HiPure Filter Column protocol reduces time and effort for plasmid purification. The kits are designed to efficiently isolate plasmid DNA from E. coli in 1.5–2.5 hours using anion-exchange columns without the use of any organic solvents or cesium chloride (CsCl). The isolated plasmid DNA purity is equivalent to two passes through CsCl gradients and has low endotoxin levels.
主要试剂
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 Maxi Columns
Column Holder
PureLink™ HiPure Precipitator Module (supplied with cat. nos. K2100-26 and K2100-27 only)
Note: For Maxipreps of low copy number plasmids from bacterial cultures of >200 mL, use twice the amount of Resuspension Buffer (R3), Lysis Buffer (L7), and Precipitation Buffer (N3) as directed in the protocol.
Order the PureLink™ HiPure BAC Buffer kit for additional buffers if the buffers in the kit are insufficient for using all of the columns when following this protocol.
实验材料
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
50-mL centrifuge tubes capable of withstanding centrifugation forces >12,000 × g
Centrifuge capable of centrifuging at >12,000 × g at 4°C
实验步骤
1. Before Starting
Verify that the Resuspension Buffer (R3) contains RNase A, and no precipitate has formed in the Lysis Buffer (L7).
2. Equilibrating the Column
The PureLink™ HiPure Filter Maxi Columns are prepackaged with the Filtration cartridge inserted into the column housing.
1) Use the Column Holder to support a HiPure Filter Maxi Column in the mouth a flask, or place the Maxi Column on the PureLink™ Nucleic Acid Purification Rack (see manual supplied with the rack for more details).
2) Apply 30 mL Equilibration Buffer (EQ1) directly into the Filtration Cartridge, which is inserted into the Maxi Column.
3) Allow the solution in the HiPure Filter Maxi Column to drain by gravity flow.
4) Prepare the cell lysate (see below) while the HiPure Filter Maxi Column is equilibrating.
3. Preparing Cell Lysate
1) For high copy number plasmids, use 100–200 mL of an overnight LB culture per sample. For low copy number plasmids, harvest 250–500 mL of an overnight LB culture per sample.
2) Harvest the cells by centrifuging the overnight LB culture at 4,000 × g for 10 minutes. Remove all medium.
3) Add 10 mL Resuspension Buffer (R3) with RNase A to the pellet and resuspend the cells until homogeneous.
4) Add 10 mL Lysis Buffer (L7). Mix gently by inverting the capped tube until the lysate mixture is thoroughly homogeneous. Do not vortex. Incubate at room temperature for 5 minutes. Note: Do not allow lysis to proceed for more than 5 minutes.
5) Add 10 mL Precipitation Buffer (N3) and mix immediately by inverting the tube until the mixture is thoroughly homogeneous. Do not vortex.
6) Proceed to Loading Filter Column and Washing DNA
4. Loading Filter Column and Washing DNA
1) Transfer the precipitated lysate from Step 5 in Preparing Cell Lysate including all the precipitated material into the equilibrated HiPure Filter Maxi Column. Let the lysate run 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.
2) Optional: The final DNA yield may be increased by washing the residual bacterial lysate in the HiPure Filter Maxi Column with 10 mL Wash Buffer (W8). Again, let the buffer flow through the HiPure Filter Maxi Column by gravity flow until the flow stops or dripping becomes very slow.
3) Immediately after the HiPure Filter Maxi Column has stopped dripping, remove the inner Filtration Cartridge from the column and discard. Note: Use the HiPure Filtration Cartridge only once. The cartridge is for single use only.
4) Wash the Maxi column with 50 mL of Wash Buffer (W8). Allow the solution in the column to drain by gravity flow. Discard the flow-through.
5) Proceed to Eluting DNA, (below).
5. Eluting DNA
1) Place a sterile 50-mL centrifuge tube (elution tube) under the HiPure Filter Maxi column.
2) Add 15 mL Elution Buffer (E4) to the Maxi 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 Maxi column.
4) Proceed to Precipitating DNA with Isopropanol, (below) or Precipitating DNA Using Precipitator Module
6. Precipitating DNA with Isopropanol
1) Add 10.5 mL isopropanol to the DNA to the elution tube. Mix well.
2) Centrifuge the tube at >12,000 × g for 30 minutes at 4°C. Carefully remove and discard the supernatant.
3) Add 5 mL 70% ethanol to resuspend the DNA pellet.
4) Centrifuge the tube at >12,000 × g for 5 minutes at 4°C. Carefully remove and discard the supernatant.
5) Air-dry the pellet for ~10 minutes.
6) Resuspend the DNA pellet in 500 μL TE Buffer (TE). For low copy number plasmids, use 200 μL TE Buffer (TE).
7. Storing DNA
Store the purified DNA at –20°C, or proceed to desired downstream application.
注意事项
1. DNA precipitation can be completed using centrifugation or with the PureLink™ HiPure Precipitator Module (included with the FP Maxiprep Kit or purchased as a separate kit). The precipitator module allows DNA precipitation within 10 minutes without any centrifugation steps. Refer to the section below to precipitate DNA with isopropanol by centrifugation. For DNA precipitation using the PureLink™ HiPure Precipitator Module. For a detailed protocol on using the precipitator module, refer to the product insert included with the precipitator.
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 at room temperature for 1 minute. 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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