PurificationofGenomicDNAUsingPureLink™SilicaColumns

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