实验概要
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.
主要试剂
Sterile, DNase-free 1.5 ml microcentrifuge tubes for elution
Sterile water, pH 7-8.5, if you are using water for elution
Wash Buffers (W4) and (W5)
Elution Buffer (E1)
主要设备
Microcentrifuge capable of centrifuging >10,000 x g
PureLink™ Spin Cartridge in Collection Tubes
Wash Tubes
实验材料
Lysates prepared
实验步骤
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
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 (page 13) 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. ntrifuge 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.
4. 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.
5. 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
1. Store the purified DNA at -20° C or use DNA for the desired downstream application.
2. 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.
3. 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.
注意事项
Follow the recommendations below to obtain the best results:
1. Perform all centrifugation steps at room temperature
2. Perform a 1 minute incubation step with Elution Buffer (E1) or water
3. Be sure to perform the recommended wash steps to obtain the best results
4. Always use sterile water, pH 7-8.5, if you are using water for elution