Year: 2020
Type: article
Abstract: Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-inoil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences.However, the recovery of ePCR products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns, making the overall process cumbersome.In this benchmark, we have described a quick ePCR extraction protocol for the purification of ePCR products, which directly employs silica-based DNA purification columns; products purified using this method have been found to be compatible with gene cloning and next-generation sequencing applications.The method described here makes ePCR easy, safe and within the reach of every laboratory. METHOD SUMMARYHere we describe a simplified method for direct breakage of the emulsion using the DNA binding buffer (containing guanidine hydrochloride and isopropanol) that is provided with commonly used silica column-based purification kits, followed by purification of the PCR products using silica columns.This method eliminates the routinely performed multi-step breakage of emulsion and extraction of DNA using hazardous organic solvents, making it simpler and safe for use in any laboratory.
Source: BioTechniques
Authors: Vaishali Verma, Amita Gupta, Vijay K Chaudhary
Institution: University of Delhi
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,415
OpenAlex ID: W3020748469