Amplicon Purification
Amplicon Purification - Related Videos
08:57
Amplicon Sequencing using the Long-Read Sequencing Technologies
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2025
This protocol was optimized for targeted deep sequencing of 18 drug-resistance regions in Mycobacterium tuberculosis using a long-read sequencing platform, followed by analysis with a tuberculosis-specific bioinformatics pipeline designed for long-read data.
10:24
Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
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Cited by 119 •
2014
Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.
08:16
Plasmid Purification
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2023
Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study. In this video, a step-by-step generalized procedure is described for how to perform plasmid purification.
03:29
Capillary Electrophoresis: A Technique to Analyze Fluorescently Labeled Polymerase Chain Reaction Amplicons
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2025
This video describes the technique of capillary electrophoresis to analyze fluorescently labeled PCR amplicons obtained from various samples based on their size. It generates a migratory pattern that determines the fragments’ base-pair lengths with the help of the labeled fluorescent dye in each fragment.
06:30
Gel Purification
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2023
Gel purification is used to recover DNA fragments after electrophoretic separation. DNA recovery from an agarose gel includes three basic steps: binding, washing and eluting from a silica column. DNA is believed to bind to silica in the presence of high salt via a salt bridge. Following binding, DNA is washed of impurities and eluted under low salt conditions disrupting this interaction. This video goes through a step-by-step, generalized procedure for cutting out a band from the gel, gel...
FAQs
Silica membrane columns and magnetic beads use differences in DNA binding behavior to retain amplicons while residual primers, free nucleotides, enzymes, and salts are removed. The target DNA is then recovered from the purification system in a cleaner form. This selective handling produces a template better suited to later enzymatic and analytical steps.
A PCR mixture may contain the desired amplicon alongside nonspecific DNA products. When the target must be distinguished from products of different sizes, gel extraction provides size-based separation before recovery. Column or bead methods are more appropriate when the main objective is to clean the amplification mixture without isolating a target band by size.
The choice depends on the type of separation required. Silica columns and magnetic beads support cleanup based on DNA binding properties, whereas gel extraction separates fragments by size. Therefore, a simple cleanup can use a column or beads, while a mixture containing an unwanted product that differs in size may call for gel extraction.
A typical workflow begins by identifying the amplified DNA that must be retained and selecting a compatible purification approach. The PCR mixture is processed through a silica column, magnetic-bead system, or gel-based size separation, and the recovered amplicon is carried forward as a cleaner template. The resulting DNA can then enter sequencing, cloning, or genotyping workflows.
Purification reduces carryover of primers, free nucleotides, enzymes, salts, and nonspecific products that can interfere with later enzymatic reactions. Removing these materials improves the suitability of the DNA template for downstream analysis. In practice, cleaner amplicons support more reliable and reproducible results in sequencing, cloning, genotyping, and related workflows.
This cleanup is especially useful after PCR when the amplified DNA will serve as input for another molecular biology procedure. Sequencing requires a cleaner template for analysis, cloning benefits from isolated amplification products, and genotyping depends on dependable DNA inputs. Purification therefore connects amplification with several common biological workflows while reducing contamination-related variability.