Mastering Molecular Cloning: a Guide to Polymerase Chain Reaction (PCR) Protocol

Polymerase chain reaction (PCR) is a molecular biology technique that clones and amplifies specific regions of DNA. PCR is a way to copy the naturally occurring cellular process of replication that occurs before a cell divides. This is an in vitro method, meaning that it is performed in test tubes in a lab rather than within a living organism (which would be in vivo). 

PCR has three stages that all work together to clone and amplify DNA: denaturing, annealing, and extending. Each stage is characterized by a different temperature. Denaturation occurs at 94-95°C, and it is when the two strands of DNA separate. Annealing occurs between 55-65°C, and this is when primers bind to the target sequence. Extension occurs at 72°C, and this is when the new copies of DNA are made. Each stage is repeated in order between 30 and 40 times to achieve the final product. Each cycle doubles the amount of DNA, so 30 cycles form over a million copies of the desired segment. 

PCR products have a wide range of applications. One of its main uses is to test for the presence of an illness (think of a PCR COVID test). A common follow-up experiment is gel electrophoresis, which sorts the DNA segments by size for visualization. Other common techniques include sending the product for sequencing or inserting the region into a plasmid for cloning. PCR also has forensic applications, wherein they can compare DNA samples from evidence and match them to a suspect; this is also how doctors perform paternity tests. PCR is a very versatile technique that has applications that span across different fields and disciplines of daily life.

• Gloves
• PCR tube strip
• 0.5-10μL pipette
• 10μL tips
• 2-20μL pipette
• 20μL tips
• Forward primer
• Reverse primer
• Template DNA
• DI water
• Polymerase mastermix
• Centrifuge 
• PCR machine

When working with DNA, it is important to limit any contaminants. Our hands hold a lot of DNA and germs that could ruin the results, so it’s best to keep them covered.

One PCR experiment will take up two tubes of a PCR tube strip: one tube for the experimental sample, and the other for the control. Control samples are important to make sure that the experimental sample isn’t contaminated.

Primers are sets of DNA that are similar to the DNA segment that we want to clone, and they are what allow us to amplify the cloned DNA. Both a forward primer and a reverse primer are needed to achieve this. Using the 0.5-10μL pipette and 10μL tips, place 1μL of the forward primer into both the experimental and control tubes. Then, place 1μL of the reverse primer into the same tubes.

• It’s important to discard and reapply new pipette tips in between each sample to limit contamination.

Template DNA is the genetic code that hosts the segment that we want to clone. Using the 0.5-10μL pipette and 10μL tips, place 1μL of the template DNA into the experimental tube only.

• We don’t add it to the control sample in order to see if there’s any DNA in the primers that amplifies even without the template DNA.

DI stands for deionized, meaning that DI water is free of any minerals or ions that could disrupt the results. In this experiment, DI water is mainly used as a buffer to fill the space in the tube. In the experimental tube, using the 2-20μL pipette and 20μL tips, add 9.5μL of DI water. In the control tube, add 10.5μL of DI water.

• The extra 1μL in the control sample makes up for the lack of template DNA from the previous step.

Polymerase mastermix is a solution that contains all of the molecular tools and enzymes to clone and amplify the DNA. Using the 2-20μL pipette and 20μL tips, add 12.5μL of polymerase mastermix to each of the experimental and control tubes. There should now be a total of 25μL in both tubes.

Centrifuging is an important step to make sure that our samples are all collected at the bottom of the tubes so that they can interact accordingly. Centrifuging too much can cause our DNA to unravel, so it’s recommended to only centrifuge for around 3-5 seconds.

• When it comes out of the machine, check to make sure that there’s no leftover solution at the top of the tubes.

PCR machines are called thermal cyclers, and what they do is cycle through the different stages and temperatures of PCR over and over again. My lab has the Veriti Dx 96-well Thermal Cycler, so these are the exact instructions for this model, but the steps for other machines should be similar. 

• 8.1: Place the tube strip into the machine. 
• 8.2: Close the lid.
• 8.3: Lock the lid. Locking the lid is a crucial step. If the lid isn't locked, then all of the heat can escape and the machine won’t be able to cycle through the stages. 
• 8.4: Click “Browse/New Methods”.
• 8.5: Click “Hifi polymerase”.
• 8.6: Run the experiment. Adjust the “Step 2” temperature if needed. This is the annealing stage, and this is the only temperature that should be altered. When the annealing temperature is set, click “Run” to start the experiment. It should run for approximately 1.5 hours.

Once the run has ended, unlock the lid and remove the tube strip. The solutions in the tubes should look identical to how they did when they went in, but the finished product will be cloned and amplified DNA (in the experimental tube only).

Woohoo! You just cloned DNA!