Cosmid DNA Isolation

The PureLink HiPure Plasmid Purification Kits allow isolation of high yields of highly pure cosmid DNA. 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 cosmid 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 cosmid DNA using different starting culture volumes.

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:

• 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

Downstream 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
 

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 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.

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/mLl 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.

Preparing cell lysate

1. Harvest 3 ml cells by centrifuging at 9,000 x g for 15 minutes.  Thoroughly remove all medium.


2. Add 0.4 ml Resuspension Buffer (R3) containing 100 µg/ml RNase A to the pellet and resuspend the cells until homogeneous.  Transfer cell suspension to a centrifuge tube.


3. Add 0.4 ml Lysis Buffer (L7).  Mix gently by inverting the capped tube five times.  Do not vortex.  Incubate at room temperature for 5 minutes.


4. Add 0.4 ml Precipitation Buffer (N3) and mix immediately by inverting the tube until the mixture is homogeneous.  Do not vortex.


5. Centrifuge the mixture at >12,000 x g at room temperature for 10 minutes. 

Note:

 
Binding and washing DNA

1. Pipette the supernatant from Step 5 onto the equilibrated column.  Allow the solution in the column to drain by gravity flow.


2. Wash the column twice with 2.5 ml Wash Buffer (W8).  Allow the solution in the column to drain by gravity flow after each wash.  Discard the flow-through.

 
Eluting and precipitating DNA

1. Place a sterile centrifuge tube (elution tube) under the column.


2. Add 0.9 ml 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.


3. Add 0.63 ml of isopropanol to the elution tube.  Mix and place on ice for 10 minutes.


4. Centrifuge the mixture at >15,000 x g at 4°C for 20 minutes.  Carefully remove and discard the supernatant.

Note:  


5. Resuspend the DNA pellet in 1 ml 70% ethanol.


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


7. Air-dry the pellet for 10 minutes.


8. Resuspend the DNA pellet in 50 µl TE Buffer (TE).

 
Note:    If a fixed-angle rotor was used for the centrifugation, the pellet will be spread over the tube walls.  Make sure to wash off the tube walls when resuspending the pellet.
 
Expected results

This procedure allows purification of a ~45 kb cosmid DNA with yields of ~4 µg DNA per 3 ml culture.

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 260 nm 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) = A_²⁶⁰x 50 x dilution factor
For DNA, A_²⁶⁰ = 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 A_²⁶⁰/A_²⁸⁰ 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.