The polymerase chain reaction (PCR) is a technique that creates millions of copies of a specific DNA sequence, and the resulting material is called the PCR product. Following the amplification process, it is standard practice to use Agarose Gel Electrophoresis to analyze this product. This technique separates the DNA fragments based on size, which allows researchers to verify that the target sequence was successfully amplified and confirm its expected length. Obtaining a clear and distinct band on the gel is entirely dependent on loading the appropriate amount of DNA. Loading too little DNA results in an invisible or faint band, while loading too much can lead to poor separation and smearing.
Assessing PCR Product Concentration
Determining the volume to load requires knowing the DNA concentration in the reaction tube, which is measured in nanograms per microliter (ng/µL). The most common quantitative method is UV spectrophotometry, often using a NanoDrop, which measures the sample’s absorbance at 260 nanometers. This measurement allows for a quick calculation of the total nucleic acid concentration, providing a numerical value for the DNA yield.
However, spectrophotometry measures all molecules that absorb UV light at that wavelength, including leftover primers, free nucleotides, RNA, or chemical contaminants like phenol. Since it cannot distinguish between the desired PCR product and these other components, the calculated concentration may be artificially inflated.
To obtain a more specific estimate of the functional PCR product, a semi-quantitative method uses an analytical gel. This involves running the sample alongside a DNA mass ladder, which contains fragments of known size and mass. By visually comparing the intensity of the sample band to the known mass of a nearby ladder band, researchers can estimate the amount of DNA present in the volume loaded. This visual comparison method is often more reliable for assessing the concentration of the specific target band, especially when the sample is unpurified.
Key Variables Influencing Optimal Load Mass
The optimal amount of DNA to load is governed by the target mass in nanograms (ng), not merely the volume in microliters (µL). For most routine analytical purposes, a target mass between 50 and 150 ng of PCR product is sought to produce a bright and clearly defined band. The specific mass required is adjusted based on the sensitivity of the detection system used to visualize the DNA.
The most influential variable is the type of fluorescent DNA-binding stain incorporated into the gel or applied after electrophoresis. Less sensitive, traditional stains, such as Ethidium Bromide, may require a higher mass of DNA, needing 10 to 20 ng to be visible. Conversely, newer, more sensitive dyes like SYBR Green or GelRed can detect a much smaller amount, providing a visible band with as little as 1 to 5 ng of target DNA. The chosen stain dictates the minimum mass threshold for a successful result.
The physical properties of the gel also affect the required mass for visualization. Gels with a higher percentage of agarose or greater thickness can scatter more light, potentially requiring a higher DNA mass for the band to appear with the same intensity. Additionally, the size of the amplified fragment influences the required mass. A longer DNA fragment needs a greater mass compared to a short fragment to achieve the same brightness because the fluorescent stain binds along the entire length of the molecule.
Calculating and Loading the Final Volume
Once the target load mass is determined and the PCR product concentration is secured, the final volume to load onto the gel can be calculated. The practical formula is: desired load volume (µL) equals the target load mass (ng) divided by the sample concentration (ng/µL). For instance, if the target mass is 50 ng and the measured concentration is 20 ng/µL, the required volume of the PCR product is 2.5 µL.
This calculated volume is then mixed with loading dye. The dye serves two primary functions: it adds density to the sample, ensuring it sinks into the well, and it contains tracking dyes that migrate through the gel, allowing the user to monitor the electrophoresis progress. A total volume of 5 to 10 µL is typically loaded into the well, which includes the calculated volume of the PCR product plus the loading dye.
Underloading the well results in a faint or invisible band, failing to meet the stain’s detection threshold. Conversely, overloading with too much DNA mass causes the band to appear as a poorly resolved smear or a distorted, thick band, as the high concentration of DNA overwhelms the separation capacity of the gel matrix. If the sample concentration is very high, it is advisable to dilute the product before calculating the final volume to prevent smearing and ensure accurate band resolution.