How to Separate DNA From White Blood Cells
Deoxyribonucleic acid, or DNA, is found in every part of a living organism. It affects every aspect of the way you develop, from the color of your hair to your emotional nature. DNA is found in the nucleus of a cell; since mature red blood cells have no nucleus, DNA must be taken from either white blood cells (leukocytes), or immature and nucleated red blood cells. DNA extraction methods are universal regardless of the source of the material--cheek scrapings, tissue biopsies, or hair, for example--but some particular processes are necessary when working with blood, due to its delicate nature. The following protocol requires prior training in basic laboratory techniques, understanding of precautions and laws pertaining to the handling of infectious biologics.
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Things You'll Need
- Required Materials:
- 10-50ml of peripheral blood collected in anticoagulant (e.g. EDTA)
- Tabletop centrifuge (e.g. Beckman TJ-6 centrifuge)
- 1ml centrifuge tubes
- Pipettes and automatic pipettes (e.g. Pipette-Aid)
- Filter pipette tips and serological pipettes
- Weighing scales
- 15ml Falcon tubes
- Tube racks
- Biohazard waste bins, bags, bag ties and labels
- Required Reagents
- 1X Phosphate-buffered saline (PBS)
- Ethanol
- Phenol/Chloroform
- Tris-EDTA (TE buffer)
- RNAse A
- Red blood cell lysis solution (1 mM EDTA, 10 mM Tris-HCl, pH 7.8, 10 mM NaCl, 1% SDS and 100µg/ml proteinase-K). This is to be heated to 55°C before starting the extraction.
Instructions
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Genomic DNA Extraction From Peripheral Blood Leukocytes:
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1
Isolate the leukocytes. Transfer the blood to a V-bottomed centrifuge tube such as a 15-milliliter Falcon tube. Centrifuge the sample for 10 minutes at 300g or 1200rpm to pellet the cells from the plasma. Discard or freeze the plasma if this is needed. The thin layer in between the pelleted red blood cells and the plasma is the buffy coat, which contains white blood cells, from which DNA will be extracted. Very carefully aspirate this layer and transfer to a fresh tube.
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2
Perform cell lysis to release DNA. Resuspend the buffy coat in 10 to 20 milliliters of pre-warmed lysis solution and gently rotate at 55 degrees Centigrade for 2 to 3 hours.
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3
Clean up. Extract the resulting lysate 3 to 4 times by resuspending it in phenol/chloroform under a chemical laminar flow hood, following the supplied instructions for the phenol/chloroform (may vary for different manufacturers). Each time, collect only the upper phase and discard the lower phase. Follow this with either an ether or chloroform extraction step. The final preparation should be void of any hemoglobin.
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4
Precipitate. Using ethanol, extract the DNA from the solution, then resuspend the pellet in an appropriate volume of TE (this is usually judged by eye depending on the size of the pellet and can very from a few microliters to a full milliliter). Add 20 micrograms per milliliter of RNAseA for an hour to inactivate RNAses.
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5
Purify. Repeat the phenol/chloroform extraction and ethanol precipitation steps three more times, and resuspend in a final volume of up to 500 microliters of TE buffer. Read the purity of this preparation on a spectrophotometer. Ideally, an aborbance at 260/280 of 1.6-1.8 is desired; otherwise repeat Step 5.
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1
Tips & Warnings
The buffy coat sits very lightly on top of the pelleted red blood cells and must be aspirated extremely slowly and gently so as not to disrupt the red blood cell fraction.
Immediately place the DNA on ice once it has been extracted to prevent degrading.
Handling blood is considered a highly infectious procedure. Do not proceed without prior clearance from local law enforcement or regulatory officials. Always maintain a sterile environment and handle all chemical using personal protective equipment.
