The ReadiPrep™ PCR Purification Kit streamlines the cleanup process following polymerase chain reaction (PCR), which is essential for ensuring the quality of downstream applications in molecular biology. Residual components from PCR, such as primers, enzymes, and dNTPs, can hinder accurate results in high-stakes applications, including forensic DNA profiling, next-generation sequencing (NGS), DNA cloning and environmental assessments. This study highlights the efficacy of the ReadiPrep™ PCR Purification Kit, which utilizes a silica column-based method for optimal separation of PCR products from contaminants. In a rapid bind-wash-elute protocol lasting under 10 minutes, the kit demonstrated comparable yield and purity to a leading competitor's product when purifying an amplified PCR product. Gel electrophoresis further confirmed the absence of undesirable fragments, showcasing the kit's capability to maintain high-quality DNA suitable for various molecular biology workflows. The findings underline the ReadiPrep™ PCR Purification Kit as a reliable and cost-effective solution for PCR cleanup, enhancing efficiency in laboratory practices.

Polymerase chain reaction (PCR) is a fundamental technique in molecular biology. However, the quality of subsequent applications can be significantly affected by residual components from the amplification process, such as primers, enzymes, dNTPs, and other inhibitors. Therefore, effective purification and cleanup protocols are essential for various downstream applications. These include forensic DNA profiling, next-generation sequencing (NGS), environmental DNA assessments, as well as other molecular biology workflows such as cloning, labeling, in vitro transcription, and quantitative assays.

Several PCR cleanup methods and kits are available, each with its own advantages, disadvantages, cost, scalability and applications. The classical techniques, ethanol precipitation and phenol-chloroform extraction, require no special kits and are cost-effective (Gautam et al., 2022). However, they are labor-intensive, time-consuming, and often vulnerable to contamination. Additionally, phenol-chloroform is toxic and hazardous, posing disposal issues. For fast PCR purification in sequencing preparation, enzymatic cleanup is used because it is simple, quick, and has low hands-on time. However, it only removes primers and nucleotides, and not proteins and other contaminants. Commercial solutions, including silica column- and magnetic-based purification protocols, are preferred mainly by scientists as they generate high DNA yield and purity following a simple procedure. Magnetic-based purification, however, is more expensive and requires magnetic racks and solvents, which usually require method optimization. Thus, silica column-based purification is a go-to method for PCR cleanup and a standard purification technique for cloning, sequencing, and general molecular biology workflows, which is widely available in kits.

We at AAT Bioquest introduced a silica column based clean up solution, ReadiPrep™ PCR Purification Kit, which offers a more cost-effective solution compared with other commercially available kits. Its optimized formulations and proprietary buffers enable selective DNA binding to the silica membrane, efficiently separating the PCR products from contaminants and ensuring they are of high purity, suitable for downstream applications. Here, we demonstrate a rapid and convenient PCR cleanup of an amplified eukaryotic 18S rRNA cDNA using the ReadiPrep PCR Purification Kit, following its simplified bind-wash-elute protocol in under 10 minutes (Figure 1).

PCR amplification of the eukaryotic 18S rRNA sequence in cDNA samples was performed using the TaqMan™ Fast Reagent Starter Kit (Thermo Fisher Scientific) with TAQuest™ qPCR Master Mix for TaqMan Probes *No ROX* (AAT Cat# 17282) following the manufacturer’s protocol, and 20 µL reactions were run on the 7500 FAST Real-Time PCR System (Thermo Fisher Scientific). Reactions were run as follows: 95°C for 20 seconds (pre-denaturation); 40 cycles of 95°C for 30 seconds (denaturation) and 60°C for 1 minute (annealing/extension).

PCR purification of the amplified DNA was performed using the ReadiPrep PCR Purification Kit (Cat. #60511). Five volumes of ReadiPrep™ Binding Buffer were added to the PCR sample, and the mixture was transferred to the ReadiPrep™ Spin Column followed by centrifugation at 13,000 rpm for 1 minute. To remove any contaminants, 600 μL of ReadiPrep™ Wash Buffer working solution was added to the column and centrifuged at 13,000 rpm for 1 minute. To recover the purified DNA, 30 μL of ReadiPrep™ Elution Buffer was added to the center of the column and centrifuged at 13,000 rpm for 1 minute. For comparison, PCR purification was performed using a competitor’s kit according to their suggested protocol. DNA yield and purity were measured using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific). For gel electrophoresis, an equal amount of purified PCR products from the ReadiPrep™ PCR Purification Kit and the competitor’s kit was loaded in 1% TAE and run for 90 minutes at 80 V. The gel was stained with Gelite™ Safe DNA Gel Stain (Cat. #17700).

Successful PCR amplification of the 18S rRNA using the primer pair 18S-F/18S-R resulted in an amplicon of approximately 300 base pairs (bp). The PCR product was cleaned up using the ReadiPrep™ PCR Purification Kit (Cat. #60511) and a competitor’s kit. Similar yields and purities were obtained for both protocols, indicating comparable recovery from the PCR purification. A total of 200 ng purified DNA from each kit was further analyzed using gel electrophoresis (Figure 2). Intense, distinct bands were observed for both kits, indicating an equal loading concentration of the ~300 bp DNA fragment. There was no presence of smears and small fuzzy bands at the bottom of the gel, also known as primer dimers. This suggests that the PCR cleanup has removed small fragments, degraded DNA, or other contaminants.

Effective PCR cleanup must remove residual primers, unincorporated dNTPs, enzymes, and other contaminants that can inhibit subsequent processes. For example, in forensic applications, where trace DNA is often degraded and present at low concentrations, even minor contaminants compromise the reliability of DNA profiles (Aljanahi et al., 2025). Another example is in NGS, where PCR purification is necessary to minimize background noise in sequencing libraries and prevent ambiguous or mixed sequencing signals, thereby ensuring high-quality sequencing data (Bronner et al., 2019). The results of our study, which demonstrate the efficacy of the ReadiPrep™ PCR Purification Kit in removing contaminants and preserving the integrity of the target amplicon, have significant implications for these and other high-stakes applications. Generally, the criteria for an optimal PCR cleanup protocol include high recovery yield, elimination of inhibitors, and preservation of target amplicon integrity.

Here, we showed that the ReadiPrep PCR Purification Kit (Cat. #60511) is a cost-effective silica-membrane-based cleanup of PCR products >100 bp comparable to existing commercial kits. This kit effectively removes, aside from enzymes and other contaminants, primers, primer dimers, and unincorporated dNTPs, which are less than 100 bp. The entire purification procedure, which utilizes a simplified bind-wash-elute protocol, takes under 10 minutes for 2-4 samples, comprising only 5 steps and potentially 1-2 additional steps outside the suggested protocol. This can include additional incubation time for binding DNA after the binding step and/or an additional wash step. The recommended ratio of binding buffer to sample volume is 5:1, and increasing the binding buffer beyond the recommended ratio still maintains the optimal conditions for DNA to bind to the silica membrane efficiently. However, although binding buffer excess is harmless, this is generally wasteful, increasing costs without improving outcomes. For purifying PCR products less than 100 bp, the gel extraction method can be used, or the binding buffer ratio can be adjusted for the recovery of small fragments. There are also other specialized commercial kits available for this purpose. Overall, the ReadiPrep™ PCR Purification Kit represents a highly effective and cost-efficient solution for PCR cleanup. This kit significantly enhances laboratory efficiency, making it an essential tool for improving the quality of experimental results.