ssM13 DNA Isolation

The PureLink™ HiPure Plasmid Purification Kits allow isolation of high yields of highly pure ssM13 (single-stranded M13) DNA from bacteria. 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 is of high purity, equivalent to two passes through CsCl gradients, and contains low endotoxin levels.  The PureLink™ HiPure Plasmid DNA Purification Kits are available in three formats that allow you to purify plasmid DNA using different starting culture volumes.  The isolated plasmid DNA is suitable for a variety of downstream applications such as mammalian transfection, automated fluorescent DNA or manual sequencing, PCR, cloning, in vitro transcription, and restriction digestion.
 
The HiPure Technology
The HiPure technology is based on anion-exchange chromatography using a patented resin composed of small particles with a uniform pore size, providing high yields and reproducible performance. The spacer arm with increased length provides improved DNA binding efficiency. The unique patented ion-exchange moiety provides high efficiency for separation of DNA from cellular contaminants including RNA.  

 

System Overview
The PureLink™ HiPure Plasmid DNA Purification Kits use a patented anion-exchange resin to purify plasmid DNA to a level equivalent to two passes through CsCl gradients. The patented resin provides excellent capacity with fast flow rates, high resolution, high yield, and efficient endotoxin removal.
 
E. coli cells are harvested, resuspended in Resuspension Buffer (R3) with RNase, and lysed with Lysis Buffer (L7). The Precipitation Buffer (N3) is added to the lysate and the lysate is clarified by centrifugation. The cleared lysate is passed through a pre-packed anion exchange column. The negatively charged phosphates on the backbone of the DNA interact with the positive charges on the surface of the resin. The temperature, salt concentration, and pH of the solutions influence binding. Under moderate salt conditions, plasmid DNA remains bound to the resin while RNA, proteins, carbohydrates and other impurities are washed away with Wash Buffer (W8). The plasmid DNA is eluted under high salt conditions with the Elution Buffer (E4).
 
The eluted DNA is desalted and concentrated with an alcohol precipitation step. The entire protocol can be completed in 1.5–2 hours.
 
Advantages
The advantages of using PureLink™ HiPure Plasmid DNA Purification Kits are:

• Purification of all types and sizes of plasmid DNA, including BAC, bacmids, and ssM13 DNAs
• High-quality purified plasmid DNA suited for mammalian transfections
• High yield of plasmid DNA (see table below)
• Reliable performance of the purified plasmid DNA in a variety of applications

The purified DNA is ultrapure and suitable for downstream applications including those requiring the highest purity, such as:

• Transfection of mammalian cells
• Automated and manual DNA sequencing
• PCR amplification
• In vitro transcription
• Bacterial cell transformation
• Cloning
• Labeling

System Specifications

Introduction
Review the information in this section before starting. Guidelines are included for growing the cell culture and amounts of starting material for use depending on the plasmid copy number.
 
Some of the PureLink™ HiPure Plasmid DNA Purification Kit buffers contain hazardous chemicals. Always wear a laboratory coat, disposable gloves, and eye protection when handling buffers.
 
Recommendations
Follow the recommendations below to obtain the best results:

• 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  

Bacterial Cultures
Grow transformed E. coli in LB medium with the appropriate antibiotic. The bacterial culture should have a cell density of approximately 10⁹ cells/mL or an absorbance at 600 nm (A600) of 1–1.5. Use bacterial culture in transition between exponential phase and stationary phase.
 
Bacterial Cultures For BAC

• Prepare a 20-h culture of BAC containing bacteria in 2X YT medium and appropriate antibiotic. The absorbance at 600 nm of the final culture should be 5.0 ± 0.5.
• Increase NaCl concentration in Wash Buffer (W8) to 0.9 M NaCl by adding 0.58 g NaCl per 100 mL Wash Buffer (W8). Conductivity should be 72 mS. This increase in salt reduces the RNA contamination in the BAC preparation.
• Warm an aliquot of Elution Buffer (E4) to 50°C.
• Add 20 mg/mL RNase A to Resuspension Buffer (R3) to a final concentration of 400 µg/mL.
• Verify that no precipitate has formed in the Lysis Buffer (L7).  

Bacterial Cultures For Bacmid

• Inoculate a single white bacterial colony into 2 mL LB with the appropriate antibiotics. Incubate the culture at 37°C in a shaking water bath at 250 rpm for a minimum of 1 h to overnight.
• Verify that RNase A is added to the Resuspension Buffer (R3) and that the Lysis Buffer (L7) contains no precipitates.

Bacterial Cultures For Cosmid

• Inoculate a bacterial culture containing your cosmid construct in medium with the appropriate selective antibiotic and grow the bacteria for 16 h (or overnight) with 225 rpm shaking.
• Add 20 mg/mL RNase A to Resuspension Buffer (R3) to a final concentration of 100 µg/mL.

Bacterial Cultures For ssM13 DNA
Inoculate an aliquot of YT medium with 1/150 volume of lawn cells (a confluent culture of the bacterial host strain). Infect the cells with an M13 colony or a phage stock. Shake vigorously for no longer than 5 h (longer incubations may result in deletions).
 
Bacterial Culture Purification Rack
The PureLink™ Nucleic Acid Purification Rack is designed specifically for use with PureLink™ HiPure Plasmid DNA Miniprep, Midiprep, and Maxiprep Kits. The PureLink™ Nucleic Acid Purification Rack consists of a 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), and Large capacity Waste Tray for collecting waste.
 
Plasmid Type and Copy Number
The PureLink™ HiPure Plasmid DNA Purification Kits allow purification of all types and sizes of plasmid DNA, including BAC, bacmids, and ssM13 DNAs.

If possible, use a high-copy-number plasmid to obtain a good yield of plasmid DNA with a small volume of culture. High copy number plasmids typically yield >2 µg DNA/mL LB whereas low-copy-number plasmids yield <2 µg DNA/mL LB.

If you are using a low copy number plasmid, you will need to use a higher volume of cell culture, as directed in the protocol.

The table below lists the volumes of cell culture required for Miniprep, Midiprep, and Maxiprep plasmid DNA purification depending on the plasmid copy number:

Resuspension Buffer (R3)
Add RNase A to the Resuspension Buffer (R3) according to instructions on the label of the bottle. Mix well. Mark the bottle label to indicate that RNase A is added. Store the buffer with RNase at 4° C.
 
Lysis Buffer (L7)
Check the Lysis Buffer (L7) for precipitates. If present, warm the solution briefly at 37° C to dissolve the precipitate.  Verify that no precipitate has formed in the Lysis Buffer (L7)
 
Equilibrating the Column
Place the PureLink™ HiPure column on the PureLink™ Nucleic Acid Purification Rack. Apply Equilibration Buffer (EQ1) to the column. Allow the solution in the column to drain by gravity flow.

Reagents and Equipment

• The Resuspension Buffer (R3), Lysis Buffer (L7), and Precipitation Buffer (N3) provided in the kit are not used in this protocol. You will use Wash Buffer (W8), Elution Buffer (E4), and TE Buffer (TE) from the kit. You will need to prepare the following solutions. Read the protocol carefully to determine the volume of each solution you need to prepare.
• M1: 3 M NaCl, 30% (w/v) PEG 8000
• M2: 100 mM Tris-HCl, pH 8.0, 25 mM EDTA
• M3: 4% SDS
• M4: 3 M potassium acetate, pH 5.5
• Store solutions M1, M2, M3, and M4 at room temperature.
• Set a water bath or heat block to 70°C

Equilibrating the Column
Place the PureLink™ HiPure column on the PureLink™ Nucleic Acid Purification Rack. Apply Equilibration Buffer (EQ1) to the column. Allow the solution in the column to drain by gravity flow.

Preparing Cell Lysate

1. Harvest the cells by centrifugation. The ssM13 DNA is in the supernatant. Transfer the supernatant to a new, sterile tube and centrifuge again to remove all traces of bacterial cells. Transfer the supernatant to a new, sterile tube.
   
   

   	Miniprep	Midiprep	Maxiprep
   Volume of culture	1–10 mL	10–25 mL	25–100 mL



2. To each 10 mL of supernatant, add 2 mL solution M1. Mix thoroughly and incubate on ice for 15 minutes to precipitate the M13 phage particles.
3. Collect the phage particles by centrifuging the sample at >10,000 x g for 10 minutes. Discard the supernatant.
4. Resuspend the phage particles in solution M2 by pipetting up and down repeatedly.
   
   

   	Miniprep	Midiprep	Maxiprep
   Volume of M2	1 mL	3 mL	9 mL

5. Lyse the phage particles by adding solution M3. Mix thoroughly by inverting the tube several times. Incubate at 70°C for the time indicated in the table.
   
   

   	Miniprep	Midiprep	Maxiprep
   Volume of M3	1 mL	3 mL	9 mL
   Incubation time	10 min	20 min	20 min



6. Add solution M4 to the lysate. Mix thoroughly by inverting the tube five times. Centrifuge at >12,000 x g for 10 minutes at room temperature.
   
   

   	Miniprep	Midiprep	Maxiprep
   Volume of M4	1 mL	3 mL	9 mL

Binding and Washing DNA

1. Load the supernatant from Step 6, above onto the equilibrated column. Allow the solution in the column to drain by gravity flow.
2. Wash the column with Wash Buffer (W8). Allow the solution in the column to drain by gravity flow after each wash. Discard the flow-through.
   
   

   	Miniprep	Midiprep	Maxiprep
   Volume of W8	2 x 2.5 mL	2 x 10 mL	1 x 60 mL

Eluting and Precipitating DNA

1. Place a sterile centrifuge tube (elution tube) under the column.
2. Add Elution Buffer (E4) to the column to elute DNA. Allow the solution to drain by gravity flow. Do not force out any remaining solution. The elution tube contains the purified DNA. Discard the column.
   
   

   	Miniprep	Midiprep	Maxiprep
   Volume of E4	0.9 mL	5 mL	15 mL



3. Add 0.7 volumes of isopropanol to the elution tube. Mix well.
   
   

   	Miniprep	Midiprep	Maxiprep
   Volume of isopropanol	0.63 mL	3.5 mL	10.5 mL



4. Centrifuge the mixture at >12,000 x g at 4°C for 30 minutes. Carefully remove and discard the supernatant.
5. Resuspend the DNA pellet in 70% ethanol.
   
   

   	Miniprep	Midiprep	Maxiprep
   Volume of 70% ethanol	1 mL	3 mL	5 mL

    
6. Centrifuge at >15,000 x g at 4°C for 5–10 minutes. Carefully remove and discard the supernatant.
7. Air-dry the pellet for 10 minutes.
8. Resuspend the DNA pellet in TE Buffer (TE).
   
   

   	Miniprep	Midiprep	Maxiprep
   Volume of TE	10–60 µL	10–60 µL	100–400 µL

Introduction
Once you have isolated DNA, you may determine the quantity and quality of the purified DNA as described below.
 
DNA Yield
Perform DNA quantitation using UV absorbance at 260nm or Quant-iT™ DNA Assay Kits.
 
UV Absorbance

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 x dilution factor
   For DNA, A₂₆₀ = 1 for a 50 µg/mL solution measured in a cuvette with an optical path length of 1 cm.

 
Quant-iT™ DNA Assay Kits
The Quant-iT™ DNA Assay Kits provide a rapid, sensitive, and specific method for dsDNA quantitation with minimal interference from RNA, protein, ssDNA (primers), or other common contaminants that affect UV absorbance.  

The kit contains a state-of-the-art quantitation reagent, pre-diluted standards for standard curve, and a pre-made buffer. The assay is performed in a microtiter plate format and is designed for reading in standard fluorescent microplate readers. Follow manufacturer’s recommendations to perform the assay.
 
Estimating DNA Quality
Typically, DNA isolated using the PureLink™ HiPure Plasmid Purification Kit has an A₂₆₀/A₂₈₀ ratio >1.80 when samples are diluted in Tris-HCl pH 7.5, indicating that the DNA is reasonably clean of proteins that could interfere with downstream applications. Absence of contaminating RNA may be confirmed by agarose gel electrophoresis.