实验原理

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.

The advantages of using PureLink™ Genomic DNA Purification Kit are:

• Rapid and efficient purification of genomic DNA from a variety of samples such as mammalian cells and tissue,mouse tails, E. coli , and yeast
• Designed to purify high-quality DNA in less than 15 minutes after sample preparation
• Simple lysis of cells and tissues with Proteinase K without the need for any mechanical lysis
• Minimal contamination from RNA
• Reliable performance of the purified DNA in PCR, restriction enzyme digestion, and Southern blotting

实验步骤

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:

• Maintain a sterile environment when handling DNA to avoid any contamination from DNases
• Ensure that no DNases are introduced into the sterile solutions of the kit
• Make sure all equipment that comes in contact with DNA is sterile including pipette tips and microcentrifuge tubes
• Do not vortex the samples for more than 5-10 seconds at each vortexing step to avoid extensive shearing of DNA
• Be sure to mix well after addition of SDS and Binding Buffer (L3)
• To minimize DNA degradation, perform lysate preparation steps quickly, and avoid repeated freezing and thawing of DNA samples

Materials Needed
96-100% ethanol
Sample for DNA isolation
10% SDS (To prepare 1 ml 10% SDS, mix 0.5 ml 20% SDS supplied in the kit with 0.5 ml deionized water, mix well and use
Phosphate Buffered Saline (PBS) for mammalian cell lysate
Zymolase buffer
Zymolase (lyticase) enzyme for yeast lysate
Sterile, DNase-free microcentrifuge tubes
Water baths or heat blocks

Components supplied with the kit

• Lysis Buffer (L6)
• Binding Buffer (L3)
• Proteinase K (>400 units/ml)
• RNase (20 mg/ml)

Preparing Lysate from Mammalian Cells

Procedure to prepare lysate from mammalian cells is described below.

1. Set a water bath or heat block at 70°C.
2. For adherent cells (up to 5 x 10⁶ cells), remove the growth medium from the culture plate and harvest cells by trypisinization or a method of choice. For suspension cells (up to 5 x 10⁶ cells), harvest cells and centrifuge the cells at 250 x g for 5 minutes to pellet cells. Remove the growth medium.
3. Resuspend the cells from Step 2 in 180 µl PBS.
4. Optional: Add 20 µl RNase A (supplied with the kit) to the sample and incubate at room temperature for 2 minutes.
5. Add 20 µl Proteinase K (supplied with the kit) to the sample and mix well by vortexing. Incubate at room temperature for 2 minutes.
6. Add 10 µl 10% SDS (prepared from 20% SDS supplied with the kit) to the lysate and mix immediately by vortexing for 5 seconds to denature proteins. Add 200 µl Binding Buffer (L3) supplied with the kit to the lysate. Mix well by vortexing for 5 seconds. The SDS precipitates in the presence of guanidine isothiocyanate.
7. Incubate at 70°C for 10 minutes to solubilize SDS and promote protein denaturation.
8. Add 200 µl 96-100% ethanol to the lysate. Mix well by vortexing for 5 seconds.
9. Proceed to Binding DNA

Preparing Mammalian Tissue Lysate

Procedure to prepare lysate from mammalian tissues is described below.

1. Set 2 water baths or heat blocks at 55°C and 70°C, respectively.
2. Place ~25 mg of minced mammalian tissue into a sterile microcentrifuge tube.
3. Add 180 µl Lysis Buffer (L6) and 20 µl Proteinase K (supplied with the kit) to the tube. Ensure the tissue is completely immersed in Lysis Buffer (L6).
   If you are using a hard mammalian tissue such as heart, you may also need to homogenize the tissue using a tissue homogenizer prior to the addition of Proteinase K.
4. Incubate at 55°C with occasional vortexing until lysis is complete (~3 hours).
5. Optional: Add 20 µl RNase A (supplied in the kit) to lysate and incubate at room temperature for 2 minutes.
6. Centrifuge the lysate at maximum speed for 5 minutes at room temperature to remove any particulate materials.
7. Transfer the supernatant to a fresh microcentrifuge tube, add 10 µl 10% SDS (prepared from 20% SDS supplied with the kit) to the lysate and mix immediately by vortexing for 5 seconds to denature proteins. Add 200 µl Binding Buffer (L3) supplied with the kit to the lysate. Mix well by vortexing for 5 seconds. The SDS precipitates in the presence of guanidine isothiocyanate.
8. Incubate at 70°C for 10 minutes to solubilize SDS and promote protein denaturation.
9. Add 200 µl 96-100% ethanol to the lysate. Mix well by vortexing for 5 seconds.
10. Proceed to Binding DNA.

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.

1. Set 2 water baths or heat blocks at 55°C and 70°C, respectively.
2. Cut the mouse tail in 0.5 cm pieces. Place one 0.5 cm piece into a sterile microcentrifuge tube and cut the tail piece into small pieces.
3. Add 180 µl Lysis Buffer (L6) and 20 µl Proteinase K (supplied with the kit) to the tube and mix well. Ensure the pieces are completely immersed in buffer.
4. Incubate at 55°C with occasional vortexing until lysis is complete (~6-8 hours). You can also perform the lysis overnight.
5. Optional: Add 20 µl RNase A (supplied in the kit) to lysate and incubate at room temperature for 2-5 minutes.
6. Centrifuge the lysate at maximum speed for 5 minutes at room temperature to remove any hair from the lysate.
7. Transfer the supernatant to a fresh microcentrifuge tube, add 10 µl 10% SDS (prepared from 20% SDS supplied with the kit) to the lysate and mix immediately by vortexing for 5 seconds to denature proteins. Add 200 µl Binding Buffer (L3) supplied with the kit to the lysate. Mix well by vortexing for 5 seconds. The SDS precipitates in the presence of guanidine isothiocyanate.
8. Incubate at 70°C for 10 minutes to solubilize SDS and promote protein denaturation.
9. Add 200 µl 96-100% ethanol to the lysate. Mix well by vortexing for 5 seconds.
10. Proceed to Binding DNA.

Preparing E. coli Lysate

Procedure to prepare E. coli cell lysate is described below.

1. Set 2 water baths or heat blocks at 55°C and 70°C, respectively.
2. Harvest up to 2 x 109 E. coli cells by centrifugation. If you are using a frozen cell pellet, proceed to Step 3.
3. Resuspend the cell pellet in 180 µl Lysis Buffer (L6) supplied with the kit. Add 20 µl Proteinase K solution (supplied with the kit) to lyse the cells. Mix well by vortexing for 5 seconds.
4. Incubate the tube at 55°C for 15 minutes.
5. Optional: Add 20 µl RNase A (supplied with the kit) to lysate and incubate at room temperature for 2 minutes.
6. Add 10 µl 10% SDS (prepared from 20% SDS supplied with the kit) to the lysate and mix immediately by vortexing for 5 seconds to denature proteins. Add 200 µl Binding Buffer (L3) supplied with the kit to the lysate. Mix well by vortexing for 5 seconds. The SDS precipitates in the presence of guanidine isothiocyanate.
7. Incubate at 70°C for 10 minutes to solubilize SDS and promote protein denaturation.
8. Add 200 µl 96-100% ethanol to the lysate. Mix well by vortexing for 5 seconds.
9. Proceed to Binding DNA.

Preparing Lysate from Yeast Cells

Procedure to prepare lysate from yeast cells is described below.

1. Set 3 water baths or heat blocks at 37°C, 55°C, and 70°C, respectively.
2. Prepare fresh 10 ml Zymolase Buffer (1 M sorbitol, 10 mM sodium EDTA, 14 mM ß-mercaptoethanol).
3. Harvest up to 5 x 10⁷ yeast cells by centrifugation. If you are using a frozen cell pellet, proceed to Step 4.
4. Resuspend the cell pellet in 500 µl Zymolase Buffer prepared as described above. Add 15 units zymolase (lyticase) enzyme and incubate at 37° C for 1 hour to generate spheroplasts.
5. Centrifuge at 3000 x g for 10 minutes at room temperature to pellet the spheroplasts.
6. Resuspend the spheroplasts in 180 µl Lysis Buffer (L6) supplied with the kit. Add 20 µl Proteinase K (supplied with the kit). Mix well by vortexing for 5 seconds.
7. Incubate the tube at 55°C for 45 minutes.
8. Optional: Add 20 µl RNase A (supplied in the kit) to the lysate and incubate at room temperature for 2 minutes.
9. Add 10 µl 10% SDS (prepared from 20% SDS supplied with the kit) to the lysate and mix immediately by vortexing for 5 seconds to denature proteins. Add 200 µl Binding Buffer (L3) supplied with the kit to the lysate. Mix well by vortexing for 5 seconds. The SDS precipitates in the presence of guanidine isothiocyanate.
10. Incubate at 70°C for 10 minutes to solubilize SDS and promote protein denaturation.
11. Add 200 µl 96-100% ethanol to the lysate. Mix well by vortexing for 5 seconds.
12. Proceed to Binding DNA.

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.

• Lyse the sample using the Lysis Buffer (L6) and Proteinase K supplied with the kit or use specialized lysis buffer or protocols to perform lysis. You may need to optimize lysis conditions prior to DNA purification to obtain the best results for your specific sample.
• Mix the sample with Binding Buffer (L3) and 96-100% ethanol prior to loading the sample onto the column. Always maintain a ratio of 1:1:1 for Lysis Buffer:Binding Buffer:Ethanol to obtain optimal DNA binding.

A general protocol for lysate preparation can be as follows:

1. For cells, harvest cells and resuspend cell pellet in 180 µl Lysis Buffer (L6) and 20 µl Proteinase K. Incubate at 55°C until lysis is complete.
   For tissues, start with a small amount of minced tissue and add 180 µl Lysis Buffer (L6). Add 20 µl Proteinase K to the sample and mix well. Incubate at 55° C until lysis is complete.
   Based on the results obtained using this lysis protocol, you may need to optimize the lysis protocol using different buffers or increasing the amount and time of Proteinase K digestion.
   If you already have a lysate, proceed to Step 2.
2. Optional: Add 20 µl RNase A (supplied with the kit). Incubate at room temperature for 2 minutes.
3. Centrifuge the lysate at maximum speed for 5 minutes at room temperature to remove any particulate material, if needed.
4. Transfer the supernatant to a fresh microcentrifuge tube and add 10 µl 10% SDS (prepared from 20% SDS supplied with the kit) to the lysate. Add 200 µl Binding Buffer (L3) supplied with the kit to the lysate. Mix well by vortexing for 5 seconds. The SDS precipitates in the presence of guanidine isothiocyanate.
5. Incubate at 70°C for 10 minutes to solubilize SDS and promote protein denaturation.
6. Add 200 µl 96-100% ethanol to the lysate. Mix well by vortexing for 5 seconds.

Purification Procedure

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

• Lysates prepared as described above
• Sterile, DNase-free 1.5 ml microcentrifuge tubes for elution
• Microcentrifuge capable of centrifuging >10,000 x g
• Optional: sterile water, pH 7-8.5, if you are using water for elution

Components supplied with the kit

• Wash Buffers (W4) and (W5)
• Elution Buffer (E1)
• PureLink™ Spin Cartridge in Collection Tubes
• Wash Tubes
• Follow the recommendations below to obtain the best results:
• Perform all centrifugation steps at room temperature
• Perform a 1 minute incubation step with Elution Buffer (E1) or water
• Be sure to perform the recommended wash steps to obtain the best results
• Always use sterile water, pH 7-8.5, if you are using water for elution

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 as described 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

1. Add 500 µl Wash Buffer (W4) supplied in the kit to the cartridge.
2. Centrifuge cartridge at room temperature at 12,000 x g for 30 seconds. Discard flow through from the Wash Tube and place cartridge into the Wash Tube.
3. Repeat Steps 1-2, once. Discard the flow through.
4. Add 500 µl Wash Buffer (W5) with ethanol to the cartridge.
5. Centrifuge the cartridge at 12,000 x g for 30 seconds at room temperature. Discard the flow through from the Wash Tube and place the cartridge into the Wash Tube.
6. Repeat Steps 4-5, once. Discard the flow through.
7. Centrifuge the cartridge at maximum speed for 2 minutes at room temperature to remove any residual Wash Buffer (W5). Discard Wash Tube.
8. Proceed to Eluting DNA.

Eluting DNA

1. Place the spin cartridge in a sterile 1.5-ml microcentrifuge tube.
2. Add 200 µl of Elution Buffer (E1) or sterile, distilled water (pH >7.0) to the cartridge.
3. Incubate at room temperature for 1 minute. Centrifuge the cartridge at maximum speed for 1.5 minute at room temperature.

The tube contains purified DNA.

1. To recover more DNA, perform a second elution step with 200 µl Elution Buffer (E1) or sterile, distilled water (pH >7.0) using another sterile 1.5 ml microcentrifuge tube.
2. Centrifuge the column at maximum speed for 1.5 minute at room temperature.
   The tube contains purified DNA. Remove and discard the cartridge.

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

• Store the purified DNA at -20° C or use DNA for the desired downstream application.
• For long-term storage, store the purified DNA in Elution Buffer (E1) at -20° C as DNA stored in water is subject to acid hydrolysis.
• 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.

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.

Analyzing DNA Yield and Quality

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.