Troubleshooting DNA Extraction from Blood

Consider these troubleshooting tips to get your DNA extraction back on track.

No matter your intended downstream application—qPCR, next generation sequencing, Sanger sequencing, and so on—you need high-quality DNA. Whether you are new to DNA extraction from blood or a seasoned pro, issues occasionally arise, and you need to be able to identify and correct them as quickly as possible to get the DNA you need. Here are useful tips that you can consider to ensure you get high quality DNA in high yield from blood for your downstream applications

Identify the problem and the possible reasons it occurred
ProTips for DNA Extraction from Blood

Identify the problem and the possible reasons it occurred

The main issues in DNA extraction from blood include low yield and contamination, which can be detected pretty quickly after completing the protocol by measuring the concentration and purity using a spectrophotometer. Additional issues include difficulty resuspending the DNA pellet (for relevant protocols), degradation—which relates to low yield, but in this sense it refers to storage, and clogged filters (when using a spin filter).

When you start troubleshooting, take the time to review your protocol and notes to identify all the possible reasons for the issue.

Problem - Low Yield
Potential Cause	Solution
Incomplete blood cell lysis	To optimize sample lysis and homogenization, you can play around with several variables. Consider the following: Increase incubation time with the lysis buffer Increase the speed/time of agitation Use a more aggressive lysing matrix Process your samples in batches and pool them together following homogenization or purification.
The frozen blood sample thawed, enabling DNase activity	Keep frozen blood samples frozen and add Proteinase K, RNase A and Lysis Buffer directly to the frozen samples. Immediately lyse the samples and let the samples thaw during incubation.
Blood sample is too old	Use fresh, unfrozen whole blood within a week. DNA degradation increases with sample age. For samples stored at 2-8°C for over 7 days, expect yields 10-15% lower than freshly isolated blood.
Protein precipitates clogged the membrane	Digestion of whole blood samples may lead to the accumulation of insoluble hemoglobin complexes that stain and clog the membrane, leading to reduced yield and purity. Prevent the formation of hemoglobin precipitates by reducing Proteinase K lysis time. Remove protein precipitates prior to applying your sample to a spin filter by pelleting the protein precipitates via centrifuging at 12,000 × g for at least 10 minutes.
Insufficient number of blood cells in the starting sample	Increase the volume of the starting sample.
Problem - Contamination
Potential Cause	Solution
High hemoglobin content	Blood samples rich in hemoglobin (indicated by a dark red color) will occasionally remain red after lysis incubation, revealing that intact hemoglobin remains abundant. Extend lysis incubation time by 3–5 minutes to improve purity.
DNA from another sample (cross-contamination) or contaminated reagents	It is often difficult to distinguish low-level contamination with another DNA sample from a true mixture of DNA templates. Use controls (positive and negative) to confidently use samples and detect contamination as early as possible. Contamination can be detected based on the results from a positive control template. The presence of unexpected peaks in the negative or positive control reactions can indicate DNA contamination. Use a designated set of equipment and reagents for DNA extraction, and thoroughly clean your workspace and equipment prior to starting.
DNA from the user (self)	Establish and perform proficiency tests and pilot experiments in the lab to ensure that contamination is a rare event. If you are in a lab that determines the DNA profile of their analysts, use a negative control and check to see if your DNA profile shows up.
Problem - Difficult to resuspend DNA
Potential Cause	Solution
DNA samples were overdried	Air dry the samples to avoid overdrying. If you’re having trouble resuspending your pellet, try heating the pellet in rehydration buffer (10 mM Tris, 1 mM EDTA, pH 7–8) at 55ºC-65°C for about 5 minutes (do not exceed 1 hour). If you are using a vacuum to dry the sample, do not exceed 5 minutes and do not use heat.

FastDNA™ SPIN Kit, 100 preps

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ProTips for DNA Extraction from Blood

ProTips for Storage

The success of your DNA extraction from blood starts with how you store your samples.

Use a DNA preservative. You can add DNA stabilizing reagents to your blood sample immediately after isolation to inhibit nuclease activity and potential contaminating microorganisms—allowing you to store unprocessed blood for extended periods of time without worrying about DNA degradation.
Minimize light exposure. Avoid exposing your sample to sunlight. UV light harms DNA by causing thymine dimers to form in DNA.
Keep your samples cool. Damaging chemical reactions within your sample increase with temperature—the faster you can refrigerate or freeze your sample, the better. If you plan on using your sample within three days, store it at 4ºC, otherwise, freeze and store your samples at -80ºC.
Add an anticoagulant. From a DNA yield and quality perspective, EDTA is the optimal choice, followed by sodium citrate. Avoid using heparin because it is difficult to remove and can interfere with PCR.
Protect your enzyme reagents. Use aliquots of enzyme-containing buffers to prevent protease degradation caused by excessive freeze-thaw cycles.

ProTips for Getting Started

Choose the best DNA extraction method for your specific experiment and lab. Common and effective options include phenol-chloroform extraction, magnetic bead separation, and precipitation chemistry. Learn more about how to extract DNA from blood.
Consider automating. There are several benefits to automating DNA extraction: more consistency between each sample, human error is eliminated, and it saves manual working time. While upfront costs can be high, the time- and cost-savings can be huge—particularly if you are processing an abundance of samples.
Do a pilot experiment. Taking the time to run through a new technique on a small subset of samples will help you optimize your protocol and identify pitfalls to avoid—making the process smooth and effective when you move forward with the full experiment.
Count your cells. Check the cell count of the sample being processed using a hemocytometer or with an automated cell counter. Too few cells will result in a low yield, and too many cells can clog filters or reduce lysis efficiency—resulting in contamination or processing issues. For high cell counts, you can split the sample into two aliquots and perform extraction separately.

ProTips for When You’re “Done”

Quantitate your DNA properly. Use a combination of spectrophotometry and agarose gel electrophoresis to visualize and quantify DNA before starting costly and time-consuming and downstream applications.
Aliquot your purified DNA. Rather than keeping your purified DNA in a single tube, distribute into individual tubes to avoid contamination and freeze-thaw cycles of your entire DNA sample.

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Sample Preparation Brochure

Quickly understand the most suitable option for your sample using this guide, which describes MP Bio’s wide selection of high-quality lysing matrices and bead beaters.

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