In science, every step has a story.
Whether you’re tweaking the temperature in a PCR cycle or measuring out just the right amount of enzyme, each action has a purpose and a reason why it’s done that way. In the lab, understanding these “whys” is just as important as knowing and correctly performing the steps. When you grasp the underlying biology, you gain confidence in your technique, develop critical troubleshooting skills, and can intelligently modify protocols for better outcomes. This deeper understanding also enhances your ability to interpret results. Beyond practical benefits, knowing the biological why’s lets you ‘see’ how you’re actively engaging with the unseen world of molecular biology – transforming routine protocols into explorations.
In this post, we’re taking you behind the scenes of our popular VNTR Human DNA Typing experiment, breaking down the rationale and mechanisms behind every step of Module I where experimenters isolate DNA from human cheeks cells. If your scientific curiosity has ever left you wondering why you swirl, heat, or spin a cell sample, you’re in the right place! And if you’re also ready to level up your DNA extractions and achieve the sharpest PCR results on the block, then it’s time to read on!
Step 1: LABEL an empty 1.5 mL screw top microcentrifuge tube and a cup of saline with your lab group and/or initials.
Why? In DNA extractions and PCR, a large number of samples are often processed simultaneously. Labeling ensures that samples are identifiable and prevents mix-ups.
Step 2: RINSE your mouth vigorously for 60 seconds using 10 mL saline solution. EXPEL the solution back into the same cup.
Why? The salt in the solution acts as a mild exfoliant, helping to break down the connections between the cells lining the cheek, which allows them to detach and enter the solution. Additionally, saline provides a balanced environment that mimics the salt concentration of the human body, helping the collected cells maintain their proper size and pressure.
Step 3: SWIRL the cup gently to resuspend the cells. TRANSFER 1.5 mL of the cell solution into the tube with your initials.
Why? Cells can quickly settle at the cup’s bottom, swirling helps to evenly disperse them throughout the solution. The 1.5 mL collection volume is the maximum safe volume for a standard micro-centrifuge tube to handle.
Step 4: CENTRIFUGE the cell suspension for 2 minutes at full speed to pellet the cells. POUR off the supernatant, the liquid above the cell pellet, but DO NOT DISTURB THE CELL PELLET!
Why? Centrifuging creates a downward force that pushes the cells to the bottom of the tube, concentrating them in the pellet. This helps collect the cells and allows for the easy removal of the saline solution.
Step 5: REPEAT steps 3 and 4 once more.
Why? (This explanation is longer but packed with important information that’s worth the read.) A key process in any PCR experiment is optimization – determining the best reaction conditions to maximize amplification and accuracy. One of the most important factors to optimize is the starting amount of cells/DNA. When DNA concentrations are too low, PCR efficiency dramatically decreases as primers struggle to find their target sequences. This can lead to faint and hard to read amplification bands or even incorrect banding patterns when primers bind to themselves! However, too many cells and too much DNA can also lead to poor or inaccurate amplification. This is because the extra DNA (plus contaminants) can inhibit polymerase activity and cause additional non-specific binding events such as the binding of primers to sequences with partial complementarity.
To determine the optimal starting concentration, researchers often perform PCR across a range of DNA or cell concentrations and compare the results. For this particular experiment, testing showed that DNA extracted from 3 mL of centrifuged cheek cell solution produced the clearest and most reliable amplification results. Hence the rinse and repeat in steps 3 and 4.
Step 6: RESUSPEND the cheek cell pellet in 150 μL lysis buffer by pipetting up and down or by vortexing vigorously. NOTE: Ensure that the cell pellet is fully resuspended and that no clumps of cells remain.
Why? The lysis buffer is added to rupture the cells and release their DNA. The buffer contains detergents with both hydrophobic (water-repelling) and hydrophilic (water-attracting) regions that disrupt the lipid bilayer of the cell membrane. This process produces what is known as a crude lysate, containing the released DNA along with other cellular components. The lysis buffer also contains Proteinase K, which breaks down nucleases (nucleases=enzymes that degrade DNA). When cell membranes break, nucleases are also released and these can damage the target DNA unless they’re quickly inactivated. Pipetting up and down or vortexing ensures that all cells come into contact with the buffer.
Step 7: CAP the tube and PLACE it in a water bath float. INCUBATE the sample in a 55°C water bath for 5 minutes.
Why? The heat from the water bath helps the lysis buffer break open cell membranes more effectively, and the 5-minute incubation gives enough time for this to happen in most cells. The higher temperature also kicks Proteinase K into high gear, making it better at breaking down proteins – especially nucleases like DNases (Deoxyribonuclease) that could damage the DNA. This step releases and protects DNA better than an extraction protocol that lacked incubation.
Step 8: MIX the sample by vortexing or by flicking the tube vigorously for 20 seconds.
Why? During the incubation in step 7, larger components of the crude lysate (such as intercellular components, proteinase K enzymes, undigested cells, etc.) may begin to settle. Vortexing or flicking redistributes these components, ensuring they remain involved in the reactions.
Step 9: INCUBATE the sample in a 99°C water bath for 5 minutes. NOTE: Students MUST use screw-cap tubes when boiling samples.
Why? This step represents a calculated trade-off. Although brief exposure to high temperatures can damage some DNA molecules (mainly through depurination), the benefits of this heating step outweigh the risks and lead to better PCR results. One reason is that the high temperature irreversibly denatures the proteinase K. ProK was key in the extractions steps 6, 7, and 8 but during PCR it becomes a liability because it can also degrade Taq polymerase. Additionally, heating disrupt DNA’s secondary structures and remove physical DNA protections such as chromatin. Both these actions increase DNA template accessibility which further increases the likelihood of a robust amplification. Screw caps are used because the higher temperature can cause an increase of pressure inside the tubes. This can cause the more common snap-top tubes to open and potentially contaminate group samples.
Step 10: CENTRIFUGE the cellular lysate for 2 minutes at full speed.
Why? This step uses centrifugal force to separate the mixture based on size and density, helping to remove unwanted cell and buffer components and purify the wanted DNA. The starting cellular lysate contains everything from broken cell membranes and proteins to organelles and lysis detergents as well as the target DNA. During centrifugation, the heavy cell debris, insoluble proteins, and other large particles form a pellet at the bottom of the tube. Meanwhile, DNA, although a large molecule, is highly soluble in water due to the negative charges of its phosphate backbone. This allows the DNA to stay in the upper liquid part of the solution.
Step 11: TRANSFER 50 μL of the supernatant to a clean, labeled microcentrifuge tube. PLACE the tube in ice.
Why? The supernatant (the clear liquid that remains at the top after centrifugation, containing dissolved molecules) contains your DNA. It does also contain some similar-sized soluble contaminants such as RNA, residual salts & detergents, and membrane fragments. However these impurities typically won’t prevent successful PCR amplification. Placing the tube on ice helps preserve the sample’s DNA integrity mainly by preventing chemical degradation and enzymatic degradation. It also slows bacterial growth that could contaminate samples and helps maintains the stable double-helix structure of the DNA.
Bonus stop step: The extracted DNA is now ready for Module II: Amplification of the D1S80 Locus. Alternatively, the extracted DNA may be stored in the FREEZER (-20°C) until needed.
Why? DNA can be stored in a freezer for years to even decades, depending on the conditions. In a standard -20°C freezer, that experiences some temperature fluctuations (such as when the door is opened or when the freezer itself powers on and off), the time frame is a few years. This is because this very cold temperatures significantly slow down most enzymes and chemical reactions that would otherwise degrade DNA. However, freezing does have a down side. Repeated freezing then thawing then freezing again can physically shear the DNA — breaking its long strands into smaller fragments due to ice crystal formation and the mechanical stress applied when this ice becomes water again. To prevent shearing, scientists limit exposing extracted DNA to just one or two freeze-thaw cycles. So if a sample is going to be used multiple times, it’s best to plan ahead and aliquot it (i.e distribute the solution into several smaller tubes) before freezing.
DNA extraction isn’t just a series of steps — it’s a process. In this process cells are gathered and exposed to forces that disrupt their architecture and free their contents. Meanwhile ProK enzymes systematically break down proteins that could threaten the newly freed DNA. Finally, these elegant strands of DNA—life’s blueprint—are separated from other molecules during centrifugation, and emerge in the supernatant ready to reveal their genetic secrets. Who doesn’t want to be a part of that beautiful process! Check our experiments so your classroom can! And stay with us as we share additional step by step posts about amplification, sequencing, CRISPR, and more! Because with the right perspective and knowledge, you can tackle endless biology questions – and lab challenges!
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