One-Step PCR Cleanup Kit

Introduction

PCR product purification is essential for successful downstream applications by removing enzymes, nucleotides, primers, and buffer components. There are several techniques for PCR product purification, including precipitation with PEG or phenol-chloroform extraction followed by ethanol precipitation, which is cost-effective but time-consuming and prone to DNA sample loss. Alternatively, bead or column-based purification is a widely used and effective method, although it requires a laborious bind-wash-elute process and involves toxic chemicals like chaotropic salts and ethanol, increasing the risk of carry-over and downstream application interference.

Recently, enzyme purification is an alternative method that uses exonuclease I to digest excess primers and alkaline phosphatase to dephosphorylate nucleotides in the reaction. The drawback of this method is that it cannot eliminate the polymerase, divalent ions (Mg2+), deoxynucleotides, and other components. In summary, the above PCR purification methods have the following limitations:

● Not very friendly to high-throughput processing and difficult to automate (spin column).

● Manual processing of samples can be laborious and potentially causes sample losses, lower yield, and user error.

● Chaotropic salt and ethanol are potentially carried over into the eluted DNA and inhibit polymerase amplification.

● Cannot wholly remove <50 bp DNA fragment.

● Purification protocols are tedious and time-consuming and cannot eliminate the polymerase, divalent ions (Mg2+), deoxynucleotides, and other components.

To address these limitations, Hygia introduce a novel one-step PCR purification – Hygia One-Step PCR Cleanup Kit is a specially designed kit for ultrafast and efficient purification of post PCR or other DNA reactions. The protocol is not only straightforward (one tube and one step) but also very flexible in removing different size DNA fragments by adjusting processing time, buffer’s pH, and detergent concentration. The magnetic Beads are added directly to the finished PCR reactions or other DNA reactions and mixed by a vortex mixer or pipetting to capture and remove the impurities (e.g, excess primer, dimer, adapter, salt, detergent, dNTPs, and enzyme). After mixing, the beads are magnetically removed, while the supernatant contains the purified and ready-to-run products. In just 1 minute, the purified DNA is ready for downstream applications, such as Sanger Sequencing, Restriction Digestion, Cloning, SNP Detection, or Library Preparation for NGS. The beads enable 96 samples to be processed simultaneously in less than 10 minutes.

Features and Advantages

● Simple protocol: No liquid transfer, One-tube, One-step

● Ultrafast: One-minute protocol

● Higher purity and recovery > 90% DNA

● Effective Cleanup: Removes excess primer (<100- mer ssDNA), dimer, adapter, a salt such as Mg2+, detergent, dNTPs, enzymes, and dye.

● Cost-effective: Eliminates columns, filters, laborious repeat pipetting, and ethanol

● High-throughput: Compatible with many different automated liquid handling systems