DOI: 10.6084/m9.figshare.30877332
title: Comparison of purification protocols for effective large-primer removal between rounds of PCR amplification

description: Effective polymerase chain reaction (PCR) product purification is essential for downstream applications like next-generation sequencing (NGS). While standard protocols remove short primers (20–30 nucleotides [nt]), NGS workflows require efficient removal of larger primers (40–50 nt) to prevent amplification artifacts. This study compared commercial kits (magnetic beads, silica columns, enzymatic degradation) with traditional isopropanol/ethanol precipitation and simple dilution for their ability to remove large primers from a 161-bp <i>KRAS</i> PCR product. Efficacy was assessed by a secondary PCR designed to amplify remaining primers. Optimal yield was achieved with isopropanol precipitation with NH<sub>4</sub>Ac (2.5–3.0 M) and overnight incubation at 4 to −20 °C. Interestingly, a simple 1:200 dilution showed comparable results. Among commercial kits, magnetic beads demonstrated superior primer removal, as evidenced by a substantially lower concentration of the secondary PCR product (0.3 ng/µL ± 0.23) compared to untreated samples (22.13 ng/µL ± 1.7). For NGS workflows, magnetic beads are the most effective method for removing large primers, while isopropanol precipitation and dilution offer viable, low-cost options, particularly for workflows involving multiple PCR steps. Efficient PCR product purification is critical for downstream applications like next-generation sequencing (NGS), yet standard methods often fail to remove large primers, causing sequencing artifacts. We compared the effectiveness of magnetic beads, silica columns, enzymatic degradation, isopropanol precipitation, and simple dilution for removing these problematic primers. While magnetic beads offered the most complete purification, traditional isopropanol precipitation also performed well as a cost-effective method. Notably, a simple 1:200 sample dilution emerged as a surprisingly effective and highly economical alternative. This work provides researchers with validated and optimized cleanup strategies, enhancing data reliability for workflows that depend on high-fidelity DNA, particularly those involving multiple amplification steps. This study evaluated several post-PCR primer removal methods for multi-step amplification workflows. We present an optimized isopropanol precipitation protocol and a simple 1:200 dilution as efficient and cost-effective alternatives to the magnetic bead purification standard used in NGS. We present an optimized isopropanol precipitation protocol that effectively removes large primers between PCR steps.The study validates low-cost strategies for a critical step in multi-round PCR workflows.Simple sample dilution can serve as a surprisingly effective, ultra-low-cost cleanup method.Our data challenge the assumption that expensive methods are required for multi-step PCR workflows. We present an optimized isopropanol precipitation protocol that effectively removes large primers between PCR steps. The study validates low-cost strategies for a critical step in multi-round PCR workflows. Simple sample dilution can serve as a surprisingly effective, ultra-low-cost cleanup method. Our data challenge the assumption that expensive methods are required for multi-step PCR workflows.