Primer Purification
Primer Purification - Related Videos
11:14
Primer-Free Aptamer Selection Using A Random DNA Library
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Cited by 20 •
2010
SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.
15:28
Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources
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Cited by 41 •
2009
We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.
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.
10:17
Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers
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Cited by 7 •
2017
Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.
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
Chemical synthesis can leave a mixture containing full-length primers, truncated sequences, synthesis byproducts, and other contaminants. Primer purification exploits differences among these components, including hydrophobicity, charge, or molecular size, to separate the desired full-length material. This separation reduces unwanted material and provides a cleaner primer preparation for downstream molecular biology reactions.
Purification level determines how effectively unwanted synthesis products are excluded from a primer preparation. Greater cleanup can therefore support more reliable PCR, DNA sequencing, site-directed mutagenesis, or synthetic biology experiments when reaction specificity matters. The practical choice is not simply maximum purification: accuracy and downstream performance must be weighed against cost for the intended experiment.
HPLC and polyacrylamide gel electrophoresis offer distinct purification options, but neither is universally superior based on the available information. Both can separate full-length primers from truncated sequences and related contaminants by exploiting differences in molecular properties. Method selection should reflect the primer and workflow requirements while balancing expected performance, accuracy, and cost.
A higher purification level is particularly relevant when primers are long, modified, or otherwise complex, or when a reaction requires high specificity. In these settings, removing incomplete oligonucleotides and synthesis-related contaminants can improve the performance of the primer material used in the experiment. The appropriate level still depends on the desired outcome and available budget.
After chemical synthesis, the primer mixture is subjected to a separation step, such as HPLC or polyacrylamide gel electrophoresis. The method distinguishes full-length primers from truncated sequences and other unwanted components using differences in hydrophobicity, charge, or molecular size. The resulting purified material can then support a downstream workflow requiring improved primer performance.
In bioengineering, purified primers support several workflows rather than one narrowly defined application. They can be used for PCR, DNA sequencing, site-directed mutagenesis, and synthetic biology, with the greatest value when specificity is important or the oligonucleotide is long, modified, or complex. Choosing suitable purification helps align primer quality with the demands and cost of the experiment.