How to Isolate DNA from Blood

Introduction

If you’re looking for a protocol on how to isolate DNA from blood for your next PCR, qPCR, next generation sequencing, or other downstream application, you are in the right place.  

Many methods exist, but regardless of the method you choose, you’ll follow the same basic steps:

1. Lyse the cells
2. Remove proteins, RNA, and other contaminants 
3. Recover the purified DNA

Solid-phase extraction are commonly used to isolate high-quality DNA from blood because it can make the process fast, reproducible, and scalable. It involves binding the DNA to a solid surface, such as silica spin columns, silica binding matrices, and silica-coated magnetic beads.

When To Use and Store Your Blood Sample

When deciding how to isolate DNA from blood, you also need to consider when you use your sample and how you store it. The integrity and length of genomic DNA isolated from blood is dependent on the quality of your starting material—which is highly influenced by the way you store and prepare your samples.

The plasma in whole blood is rich with nucleases, which threatens your DNA yield. As long as the leukocytes in the blood remain intact, these nucleases will not damage the gDNA. Your storage and sample preparation methods will affect the stability of the leukocytes and the DNA it holds. Below are some tips on how to maintain the integrity of your target DNA.

ProTips:

• Don’t store fresh blood samples at 4–8°C for longer than a week. When blood samples are stored at 4°C, over time, leukocytes become increasingly unstable and the DNA gets exposed to nucleases, which will reduce the size of purified gDNA.
• Avoid using same-day blood samples because they can be hard to lyse and it is difficult to get consistent gDNA purity between samples. Store fresh blood samples at 4–8°C for 2–3 days before you start your purification process.
• If you aren’t planning to use your whole blood samples within a few days, store them at -80°C to obtain excellent quality gDNA, but do not let these samples thaw before purification.

Why you should avoid thawing frozen whole blood samples:
As the blood samples freeze, ice crystals form, damaging the leukocytes and giving nucleases the opportunity to rapidly degrade the gDNA you need. Keeping samples frozen while you add the lysis components and then incubating immediately at 56°C protects the gDNA so you can obtain large gDNA fragments.

Prep Before You Start

• Store Proteinase K (and RNAse if using) at -20°C
• Chill your lysing solutions and PBS on ice
• Prepare your wash buffers as indicated in the protocol
• Set a water bath or heating block to 56°C for sample lysis
• Preheat the appropriate volume of elution buffer(s) to 60°C
• If you are preparing multiple samples, create a master mix of the lysis buffer, proteinase K and RNAse to minimize pipetting. You can omit RNase A if a low level of RNA contamination will not affect your downstream applications.
• Label your tubes to avoid mixing up your samples
• Wear gloves and clean your bench with alcohol to prevent contamination and DNA degradation

Lyse Your Samples

Mammalian Whole Blood

1. Transfer 100 µl of whole blood to a 1.5 ml microfuge tube (add cold PBS to bring the total volume to 100 µl) 
2. Add Proteinase K, RNase A and Lysis Buffer (or your prepared master mix) and immediately vortex. For frozen blood, do not thaw—add enzymes and lysis buffer (or master mix) to the frozen sample and immediately proceed
3. Incubate for 5 minutes at 56°C and vortex occasionally
4. Proceed to DNA Binding and Elution

Nucleated Whole Blood

1. Transfer 10 µl whole blood to a 1.5 ml microfuge tube.
2. Add 90 µl cold PBS and mix by vortexing
3. Add Proteinase K and RNase A and immediately vortex (do not add Lysis Buffer at the same time)
4. Add the Lysis Buffer and vortex (the solution will be viscous)
5. Incubate for 5 minutes at 56°C and vortex occasionally
6. Proceed to DNA Binding and Elution

Alternatively, you can use lysing matrices to lyse your samples. Learn more about how to choose the ideal lysing matrix.

DNA Binding and Elution

The binding and elution steps vary based on the solid phase you choose.

Silica spin columns: Multi-step centrifugation

1. Add binding buffer to your lysed sample and vortex thoroughly
2. Apply your sample to the column, being sure not to disturb the membrane, overfill the column, or touch the column
3. Centrifuge (speed and times vary by kit) the sample to trap the DNA on the membrane. Transfer the column to a fresh tube
4. Apply Wash Buffer and centrifuge at max speed (> 12,000 x g) for 1 minute so contaminants pass through the membrane—discard the flow through (repeat according to your specific protocol)
5. Transfer the column to a clean DNAse-free tube
6. Apply the preheated elution buffer (100 µl) to the membrane, incubate it at room temperature for 1 minute, centrifuge at max speed (> 12,000 x g) for 1 minute to release the DNA

Magnetic beads: Magnetic racks and pipetting

1. Mix the lysed sample with binding buffer
2. Apply the samples to the tubes (or wells) containing magnetic beads, allowing DNA to bind
3. Wash the beads with the Washing Buffer, gently agitating the sample
4. Use a magnetic rack to hold the beads in place to remove and discard the wash buffer
5. Repeat the wash steps according to your specific protocol (for instance the MPure Blood DNA Extraction Kit has three different washing buffers)
6. Apply your elution buffer to washed beads and agitate the tubes to release the DNA into the solution 
7. Place your tubes on the magnetic rack and pipette the eluent to a fresh, DNase-free tube Repeat the elution step according to your specific kit

Quantitate and Store the DNA

Spectrophotometrically quantitate the purified DNA. Use the purified DNA immediately for downstream analysis or store at -20°C avoid freezing and thawing the DNA repeatedly.

How to Isolate DNA from Blood with MP Bio

With MP Bio, you have options from budget-friendly reagents to high-throughput, automated systems. You can get the lysing matrices, reagents, spin columns and recovery tubes, and other equipment individually or you can get everything you need in a kit.

Here are some kit options:

FastDNA-96 Tissue & Insect DNA Kit, 2 x 96 Preps
Rapid, high-throughput isolation of PCR-ready genomic DNA from animal and insect samples, including whole blood.

MPure Blood DNA Extraction Kit, 48 Preps
Purify genomic DNA from whole blood or buffy coat and automate the process affordably. To use this kit, you’ll want to use this instrument: MPure-12™ Automated Nucleic Acid Purification System.

GNOME® DNA Isolation kit
Isolate high molecular weight genomic DNA from blood cells, nucleated white blood cells, and other mammalian cells and tissues.