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Purification of Genomic DNA Using |
The PureLink Genomic DNA Purification Kit allows rapid and efficient purification of genomic DNA. The kit is designed to efficiently isolate genomic DNA from mammalian cells and tissues, mouse tail, E. coli cells, and yeast. After preparing the lysates, the DNA is purified from lysates in less than 15 minutes using a spin column based centrifugation procedure.
The isolated DNA is 20-50 kb in size and is suitable for PCR, restriction enzyme digestion, and Southern blotting.
System overview
The PureLink Genomic DNA Purification Kit is based on the selective binding of DNA to silica-based membrane in the presence of chaotropic salts.
The lysate is prepared from E. coli cells, yeast cells, mouse tails, and mammalian cells and tissues. The cells or tissues are digested with Proteinase K in the presence of EDTA to inhibit DNases. A detergent (SDS) is added during lysis to aid in denaturation of proteins and in solubilizing membrane proteins. The SDS also stimulates Proteinase K activity. The lysis step is performed at 55° C for E. coli, yeast, and tissues to accelerate the digestion procedure. Any residual RNA is removed by digestion with RNase prior to binding samples to the spin column.
The lysate is mixed with Binding Buffer (L3) and ethanol to adjust conditions for subsequent DNA binding to the PureLink Spin Column. The DNA binds to the silica-based membrane in the cartridge and impurities are removed by thorough washing with Wash Buffers. The genomic DNA is then eluted in low salt Elution Buffer (E1) or water.
Advantages
The advantages of using PureLink Genomic DNA Purification Kit are:
System specifications
Starting Material: | Varies |
Binding Capacity: | ~1 mg nucleic acid |
Column Reservoir Capacity: | 700 µl |
Wash Tube Capacity: | 2.0 ml |
Centrifuge Compatibility: | Capable of centrifuging >10,000 x g |
Elution Volume: | 2 x 200 µl |
DNA Yield: | Varies |
DNA Size: | 20-50 kb |
Detailed protocols for preparing lysates from mammalian cells and tissues, mouse tail, E. coli, and yeast are described here. If none of the sample preparation protocols match the type or size of your sample, use the guidelines in Sample Preparation to develop your own protocol.
Introduction
The flow chart for purifying genomic DNA using the PureLink Genomic DNA Purification Kit is shown below.
Introduction
Instructions for preparing lysates from mammalian cells and tissues, mouse tail, bacteria, and yeast are described below.
To obtain high-quality genomic DNA, follow the guidelines recommended below.
The PureLink Genomic DNA Purification Kit buffers contain guanidine isothiocyanate. Always wear a laboratory coat, disposable gloves, and eye protection when handling buffers.
Do not add bleach or acidic solutions directly to solutions containing guanidine isothiocyanate or sample preparation waste as it forms reactive compounds and toxic gases when mixed with bleach or acids.
Sample amount
There are different protocols for preparing lysates depending on the starting material (sample). Based on your sample, choose an appropriate lysate preparation protocol from the table below.
The PureLink Genomic DNA Purification Kit is suitable for isolating DNA from a variety of samples using the recommended sample amount (see table below). If you wish to use less sample amount than the recommended amount listed in the table below, follow the appropriate protocol for the sample using the recommended volume of reagents except perform only one elution step or decrease the volume of elution buffer. Note: If you start with less amount of sample, the yield of DNA may also be lower.
If none of the sample preparation protocols match the type or size of your sample, then use the guidelines described below to develop your own protocol.
To obtain high yield of DNA and minimize DNA degradation, collect the sample and proceed immediately to sample preparation or freeze the sample in liquid nitrogen immediately after collection.
Sample | Amount |
Mammalian cells
|
1-5 x 10
6 cells (suspension or adherent cells)
|
Mammalian tissues
|
Up to 25 mg
|
Mouse tail
|
0.5 cm sections
|
E. coli cells
| Up to 2 x 10 9 cells |
Yeast cells
|
Up to 5 x 10
7 cells
|
RNase A digestion
RNase A digestion is performed during sample preparation to degrade RNA present in the sample and minimize RNA contamination in the purified DNA sample. RNA contamination also inflates the DNA content measured at 260 nm.
RNase A is supplied with the kit and an RNase digestion step is included as an optional step during sample preparation in the protocols described in this section. The option to perform RNase digestion step will depend on the sample type and RNA content of the sample.
If RNA content of the sample is minimal (e.g., mouse tail) and RNA contamination does not interfere with any downstream applications of the purified DNA, there is no need to perform the optional RNase digestion step during sample preparation. If RNA content of the sample is high (e.g., liver, kidney) and RNA contamination does interfere with any downstream applications of the purified DNA, perform the optional RNase digestion step during sample preparation.
Follow the recommendations below to obtain the best results:
Materials needed
Components supplied with the kit
Preparing lysate from mammalian cells
Procedure to prepare lysate from mammalian cells is described below.
Preparing mammalian tissue lysate
Procedure to prepare lysate from mammalian tissues is described below.
Preparing lysate from mouse tail
Procedure to prepare lysate from mouse tail is described below. Note: The sample preparation protocol may not require any RNase A treatment step as mouse tails contain low levels of RNA.
Preparing E. coli Lysate
Procedure to prepare E. coli cell lysate is described below.
Preparing Lysate from Yeast Cells
Procedure to prepare lysate from yeast cells is described below.
Guidelines for Lysate Protocol Development
If none of the lysate preparation protocols described in this manual match the type or size of your sample, use the following guidelines to develop your own lysate preparation protocol.
Introduction
The purification procedure is designed for purifying genomic DNA using a spin column-based centrifugation procedure in a total time of 10-15 minutes.
Materials needed
Components supplied with the kit
Follow the recommendations below to obtain the best results:
Elution volume
The DNA is eluted in 2 aliquots of 200 µl each to obtain higher DNA yield. The DNA recovery in the first elution is 65-80% and after second elution is >95%.
To prevent dilution of the DNA sample and also avoid contact of the spin column with the eluate, perform the two-elution steps using different tubes.
Before starting
Add 40 ml 96-100% ethanol to 10 ml Wash Buffer (W5) included with the kit. Store the Wash Buffer (W5) with ethanol at room temperature.
Binding DNA
1. Remove a PureLink Spin Cartridge in a Collection Tube from the package.
2. Add the lysate with Binding Buffer (L3) and ethanol prepared to the PureLink Spin Cartridge.
3. Centrifuge the cartridge at 12,000 x g for 30 seconds at room temperature.
4. Discard the collection tube and place the spin cartridge into a clean Wash Tube supplied with the kit.
5. Proceed to Washing DNA
Washing DNA
Eluting DNA
Based on the volume of elution buffer used for elution, the recovery of the elution volume varies and is usually >95% of the elution buffer volume used.
Storing DNA
The next step
You may determine the quality, quantity, and length of the purified DNA as described in Analyzing DNA Yield and Quality.
Genomic DNA isolated using the PureLink Genomic DNA Purification Kit is suitable for use in any downstream application of choice.
DNA yield
After purification with PureLink Genomic DNA Purification Kit, the yield of purified DNA can be estimated by UV absorbance at 260 nm or Quant-iT DNA Assay Kits.
UV absorbance
1. Measure the A260 of the solution using a spectrophotometer blanked against 10 mM Tris-HCl, pH 7.5.
2. Calculate the amount of DNA using the formula:
DNA (µg) = A260 x 50 µg/(1 A260 x 1 ml) x dilution factor x total sample volume (ml)
For DNA, A260 = 1 for a 50 µg/ml solution measured in a cuvette with an optical path length of 1 cm.
Note: Any contamination from RNA will inflate the DNA content measured at 260 nm. To avoid any interference from RNA, use the Quant-iT Kits.
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.
DNA quality
Typically, DNA isolated using the PureLink Genomic DNA Purification Kit has an A260/A280 >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.
DNA length
Genomic DNA isolated with the PureLink Genomic DNA Purification Kit is usually in the size range of 20-50 kb. To determine the exact size of DNA, perform PFGE (Pulse-Field Gel Electrophoresis) on an agarose gel.
The DNA isolated using the PureLink Genomic DNA Purification Kit is suitable for use in PFGE without ethanol precipitation or any additional steps. General guidelines for PFGE are described below. For details, refer to the manufacturer’s recommendations.
For PFGE, load 20 µl (0.5-1 µg) purified DNA/lane in 10X BlueJuice Gel loading Buffer on a 1% agarose gel in 0.5X TBE using appropriate PFGE molecular weight DNA ladders. Perform electrophoresis at 6 V/cm for 15 hours at 14° C using a switch time of 1-7 seconds. The gel is stained with ethidium bromide after electrophoresis to visualize the DNA.
Introduction
The DNA yield obtained from various samples is described below.
DNA yield
The yield of genomic DNA obtained from various samples using the PureLink Genomic DNA Purification Kit is listed below: The DNA quantitation was performed with the Quant-iT DNA Assay Kits (see above). The yield is the total yield from 2 x 200 µl elution.
Material | Amount | DNA Yield |
E. coli cells
|
1 x 10
9 |
5-15 µg
|
HeLa cells
|
1 X 10
6 |
5-10 µg
|
293F cells
|
1 x 10
6 |
2-6 µg
|
Huh-7 cells
|
2 x 10
6 |
3-10 µg
|
Mouse tail
|
0.5 cm
|
4-9 µg
|
Mouse liver
|
25 mg
|
5-8 µg
|
Note: The DNA yield varies with the sample and DNA content of the sample.
DNA quality
Genomic DNA isolated from various samples was analyzed by agarose gel electrophoresis on a 0.8% E-Gel agarose gel.
Samples on the gel are:
Lane 1 | 36.7 ng of DNA isolated from E. coli (5 x 108 cells) |
Lane 2 | Blank |
Lane 3 | 21.8 ng DNA isolated from human HeLa (1x106 cells) |
Lane 4 | 19.3 ng DNA isolated from human Huh-7 (2x106 cells) |
Lane 5 | 37.5 ng DNA isolated from human 293F (1x106 cells) |
Lane 6 | 60.3 ng DNA isolated from mouse liver (25 mg) |
Lane 7 | 69.1 ng DNA isolated from mouse tail (0.5-cm section) |
Lane 8 | Blank |
Lane 9 | 1 Kb DNA Extension Ladder (0.5 µg/lane) |
Introduction
Refer to the table below to troubleshoot any problems you may encounter with the PureLink Genomic DNA Purification Kit.
Problem | Cause | Solution |
Low DNA yield
|
Incomplete lysis
|
Decrease the amount of starting material used.
Be sure to add Proteinase K and SDS solution during lysis.
For tissues, cut the tissue into smaller pieces and ensure the tissue is completely immersed in the Lysis Buffer to obtain optimal lysis.
If incomplete lysis is observed, increase the incubation time or amount of Proteinase K used for lysis.
|
Poor quality of starting material
|
Be sure to use fresh sample and process immediately after collection or freeze the sample at –80°C or in liquid nitrogen. The yield and quality of DNA isolated is dependent on the type and age of the starting material.
| |
PureLink Spin Column is clogged
|
Make sure that the lysate is clear when the lysate is loaded on to the spin cartridge. Remove any particulate or viscous material by centrifugation prior to loading the lysate on to the spin cartridge.
| |
Incorrect binding conditions
|
Be sure to add Binding Buffer (L3) and 96-100% ethanol to the lysate prior to loading the samples on the spin cartridge. Mix the sample properly with Binding Buffer and ethanol by vortexing.
Avoid overloading the cartridge.
| |
Ethanol not added to Wash Buffer (W5)
|
Be sure to add 96–100% ethanol to Wash Buffer (W5).
| |
Incorrect elution conditions
|
Add elution buffer and perform incubation for 1 minute with elution buffer before centrifugation.
To recover more DNA, perform a second elution step.
| |
DNA is sheared or degraded
|
Avoid extensive pipetting to facilitate lysis/homogenization and repeated freezing and thawing of samples to prevent any DNA damage.
Maintain a sterile environment while working to avoid any contamination from DNases.
| |
Dark colored eluate or discolored membrane (mammalian tissue or mouse tails only)
|
Pigments from tissues bind to the cartridge matrix and co-elute with DNA
|
Be sure to add ethanol to the lysate prior to loading the lysate on to the spin cartridge. The ethanol prevents the pigments from sticking on the cartridge matrix.
Perform centrifugation of the lysate at a higher speed and longer time prior to loading the lysate on to the cartridge.
|
RNA contamination
|
--
|
Perform optional RNase digestion step during the sample preparation.
|
Inhibition of downstream enzymatic reactions
|
Presence of ethanol in purified DNA
|
Traces of ethanol from the Wash Buffer (W5) can inhibit downstream enzymatic reactions.
To remove Wash Buffer (W5), discard Wash Buffer (W5) flow through. Place the spin cartridge into the Wash Tube and centrifuge the spin cartridge at maximum speed for 2-3 minutes to completely dry the cartridge.
|
Presence of salt in purified DNA
|
Use the correct order of Wash Buffers for washing. Always wash the cartridge with Wash Buffer (W4) followed by washing with Wash Buffer (W5).
|
For Research Use Only. Not for use in diagnostic procedures.