PureLink™ HiPure Plasmid Filter Purification Kits - for Midi and Maxi preparation of Plasmid DNA

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.

Kit Formats

The PureLink™ HiPure Plasmid Filter Purification Kits are available in the following formats:

• The PureLink™ HiPure Plasmid Filter Midiprep and Maxiprep Kits allow you to purify plasmid DNA using different starting culture volumes.
• The PureLink™ HiPure Plasmid FP (Filter and Precipitator) Maxiprep Kit includes the PureLink™ HiPure Plasmid Filter Maxiprep Kit and PureLink™ HiPure Precipitator Module, and allows plasmid DNA purification including isopropanol precipitation without centrifugation within one hour.

The HiPure Technology

The HiPure technology is based on anion-exchange chromatography and uses a patented resin composed of small particles with a uniform pore size. The HiPure technology provides high yields of highly pure plasmid DNA with reproducible performance.

The spacer arm with increased length provides improved DNA binding efficiency. The unique patented ion-exchange moiety provides high efficiency separation of DNA from cellular contaminants, including RNA. Filter Columns The HiPure Filter Columns contain the Filtration Cartridge prepackaged in the DNA Binding Column. The column is fitted with anion-exchange resin (see below). This design of the HiPure Filter Column allows sample clarification by filtration and plasmid DNA binding in one combined step.


PureLink™ Column Holder

The Column Holders in the kit allow Midi and Maxi Columns to be supported in an upright position when placed in the mouth of an Erlenmeyer (or similar) flask.


PureLink™ HiPure Precipitator Module

The PureLink™ HiPure Precipitator Module, included with the PureLink™ HiPure Plasmid FP (Filter and Precipitator) Maxiprep Kit (cat. nos. K2100-26 and K2100-27), allows rapid isopropanol precipitation of the eluted DNA without any centrifugation steps. This saves time and reduces the risk of losing the DNA pellet during supernatant removal. To precipitate and desalt the DNA, isopropanol is added to the eluted DNA and then applied to the HiPure Precipitator using a large syringe. After a subsequent washing and drying step, the plasmid DNA is easily eluted from the HiPure Precipitator with TE buffer.


System Overview

The PureLink™ HiPure Plasmid Filter Purification Kits use a patented anion-exchange resin to purify plasmid DNA to a level of purity that is equivalent to two passes through CsCl gradients. The patented resin provides high binding capacity with fast flow rates, high resolution, high yield, and efficient endotoxin removal. E. coli cells are harvested by centrifugation, then resuspended in Resuspension Buffer (R3) with RNase A, and lysed with Lysis Buffer (L7). Precipitation Buffer (N3) is then added to the lysate. The lysate is poured into a pre-packed  anion-exchange column fitted with the Filtration Cartridge unit. In one simple combined step, the lysate is clarified  and the negatively charged phosphates of the DNA backbone interact with the positive charges on the surface of the anionexchange resin. The temperature, salt concentration, and pH of the solutions are optimized for efficient binding of DNA. A single column wash under moderate salt conditions using Wash Buffer (W8) removes RNA, proteins, carbohydrates and other impurities while the plasmid DNA remains bound to the resin.

The plasmid DNA is eluted under high salt conditions with the Elution Buffer (E4). The eluted DNA is desalted and concentrated by alcohol precipitation or using the PureLink™ HiPure Precipitator Module included with the PureLink™ HiPure Plasmid FP (Filter and Precipitator) Maxiprep Kit or available separately. The entire protocol can be completed in 1.5–2 hours.

Advantages

The PureLink™ HiPure Plasmid Filter Purification Kits offer the following advantages:

• Bacterial lysate clarification by a simple filter column procedure without centrifugation
• No centrifugation required for alcohol precipitation when using the PureLink™ HiPure Precipitator Module
• High-quality purified plasmid DNA suited for mammalian transfections
• High plasmid DNA yields with up to 350 μg for Midipreps and 850 μg for Maxipreps
• Reliable performance of the purified plasmid DNA in a variety of downstream applications

System Specifications

* Specifications and results are based on high copy number plasmids.
** Binding capacity depends on plasmid copy number, type and size, and volume of bacterial culture used.
*** DNA yield depends on plasmid copy number, type and size, bacterial strain, and growth conditions.


Downstream Applications

Plasmid DNA isolated using the PureLink™ HiPure Plasmid Filter Purification Kits is suitable for a wide variety of downstream applications such as:

• Mammalian transfections
• Automated fluorescent DNA or manual sequencing
• PCR
• Cloning
• in vitro transcription
• Restriction digestion.

• Midiprep kit protocol
• Maxiprep kit protocol

• Maintain a sterile environment when handling DNA to avoid any contamination from DNases.
• Ensure that no DNase is introduced into the sterile solutions supplied with the kit.
• Make sure that all equipment coming in contact with DNA is sterile, including pipette tips and tubes.
• Use the PureLink™ Nucleic Acid Purification Rack for column purification (see below).
• Perform the recommended wash steps during purification to obtain the best results.
• Use the TE Buffer (TE) provided or 10 mM Tris-HCl, pH 8.5 to resuspend the DNA pellet.

• Column Holder Rack (for processing 12 miniprep, 8 midiprep, and 4 maxiprep columns)
• Collection Tube Rack (capable of accommodating various types and sizes of recovery tubes)
• Large Capacity Waste Tray for collecting waste.

• 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

• 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

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.

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

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).

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

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).

• 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

• 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)

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.

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

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).

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

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).

• Always remove the precipitator module from the syringe before removing the plunger
• Do not apply excessive pressure while pushing the solution through the precipitator, as too much pressure may detach the precipitator from the syringe.
• Attach the precipitator to the syringe properly using the luer lock mechanism to avoid the detachment of the precipitator during sample processing.
• Always use proper aseptic techniques when working with DNA and use only sterile, DNase-free tips and tubes to prevent DNase contamination.
• When eluting the DNA with TE Buffer (TE), use a higher volume of TE Buffer to increase DNA yield. Use a lower volume of TE Buffer to increase DNA concentration (see Elution Parameters in the precipitator module product insert.
• The TE Buffer (TE) contains 10 mM Tris-HCL, pH 8.0, 0.1mM EDTA). If Tris-HCl or EDTA interferes with downstream applications, sterile water (pH 8.0) can be substituted.

1. Add 10.5 mL isopropanol to the elution tube containing the DNA (see Eluting DNA). Mix well. Incubate for 2 minutes at room temperature.


2. Remove a 30 mL syringe (supplied with the precipitator module) from the package and remove the plunger.


3. Attach the PureLink™ HiPure Precipitator through the luer lock inlet to the 30 mL syringe nozzle.


4. Load the precipitated DNA mixture into the syringe, place the precipitator over a waste container, and insert the plunger into the syringe. Use a slow, constant force to push the plunger to pass the DNA mixture through the precipitator. Discard the flow through.


5. Detach the precipitator from the syringe, remove the plunger, then reattach the precipitator to the syringe. Note: To prevent damage to the membrane, do not remove the plunger while the precipitator is still attached to the syringe.


6. To wash the DNA precipitate: Add 3–5 mL 70% ethanol into the syringe. Place the precipitator over a waste container. Insert the plunger into the syringe. Push the plunger to pass the ethanol through the precipitator.
   


7. Detach the precipitator from the syringe, remove the plunger, then reattach the precipitator to the syringe.


8. To dry the precipitator membrane: Insert the plunger into the syringe and push the plunger to pass air through the precipitator.


9. Repeat Step 8, once.


10. Blot any ethanol droplets on the precipitator nozzle with a paper towel.


11. Detach the precipitator from the 30 mL syringe and discard the 30 mL syringe.


12. Remove a 5 mL syringe (supplied with the precipitator module) from the package and remove the plunger from the syringe. Attach the precipitator to the 5 mL syringe.


13. To elute the plasmid DNA from the precipitator: Add 0.75–1.0 mL TE buffer to the 5 mL syringe. Insert the plunger, and place the precipitator over a clean, sterile microcentrifuge tube. Push the plunger to elute the plasmid DNA into the new tube.


14. Optional: Perform a second elution to maximize DNA recovery: Detach the precipitator from the syringe, remove the plunger, and reattach the precipitator to the syringe nozzle. Load the entire volume of eluate from Step 13 into the syringe. Place the precipitator nozzle over a new microcentrifuge tube. Insert and push the plunger to perform the second elution and elute the DNA into the microcentrifuge tube.


15. Store the eluted DNA at –20°C (long-term) or 4°C (short-term), or proceed to downstream application.
    

1. Prepare a dilution of the DNA solution in 10 mM Tris-HCl, pH 7.5. Mix well. Measure the absorbance at 260 nm (A₂₆₀) of the dilution in a spectrophotometer (using a cuvette with an optical path length of 1 cm) blanked against 10 mM Tris-HCl pH 7.5


2. Calculate the concentration of DNA using the formula: DNA ( μg/ml) = A₂₆₀ x 50 × dilution factor For DNA, A₂₆₀ = 1 for a 50  μg/ml solution measured in a cuvette with an optical path length of 1 cm.

Review the information below to troubleshoot your experiments with PureLink™ HiPure Plasmid Filter Purification Kits.