Cdna Purification
Cdna Purification - Related Videos
09:44
Rapid Amplification of cDNA Ends
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2023
Source: Pablo Sanchez Bosch2, Sean Corcoran2 and Katja Brückner1,2,3 1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research 2Department of Cell and Tissue Biology, 3Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA Rapid Amplification of cDNA Ends (RACE) is a technique that allows amplification of full-length cDNA from mRNA by extending to the 3’ or 5’ end, even without prior knowledge of the sequence (Frohman et al.,...
07:01
Processing the Loblolly Pine PtGen2 cDNA Microarray
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Cited by 6 •
2009
The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.
15:21
RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis
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Cited by 111 •
2009
The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.
02:35
RACE - Rapid Amplification of cDNA Ends
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2021
Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer. Since the...
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.
FAQs
High-salt conditions promote cDNA binding to either a silica membrane or magnetic beads, allowing unwanted soluble reaction components to remain available for removal during washing. The support therefore acts as a selective capture surface rather than simply concentrating the entire reverse-transcription mixture. This separation principle produces a template better suited to subsequent molecular analysis.
The cleanup removes residual RNA, primers, free nucleotides, enzymes, salts, and other components carried over from reverse transcription. These substances can contribute to background or inhibit downstream reactions, making amplification and measurement less reliable. Removing them helps PCR and quantitative PCR use a cleaner template, improving amplification efficiency, measurement accuracy, and reproducibility in gene expression studies.
A low-salt buffer releases the captured cDNA from the silica membrane or magnetic beads while limiting the carryover of salts that could interfere with later reactions. The resulting eluate provides a cleaner nucleic acid template for molecular analysis. This final exchange from binding conditions to a low-salt solution supports dependable use in amplification, cloning, sequencing, or library preparation.
Silica membranes and magnetic beads provide two common formats for the same capture-and-cleanup principle. In both cases, high-salt conditions bind the cDNA, washing removes residual reaction components, and a low-salt buffer recovers the nucleic acid. Their shared purpose is to separate the desired template from contaminants before downstream analysis, rather than to alter the cDNA itself.
The workflow begins by placing the reverse-transcription product under high-salt conditions so the cDNA binds to a silica membrane or magnetic beads. Washing then removes RNA, primers, free nucleotides, enzymes, salts, and other residual components. Finally, a low-salt buffer elutes the purified cDNA, producing a cleaner template for subsequent molecular procedures.
Purified cDNA is useful when researchers need a cleaner template for PCR or quantitative PCR, where inhibitors and background can affect amplification and measurement. It also supports cloning, sequencing, and transcriptome library preparation. The appropriate application depends on the intended analysis, but purification consistently prepares the reverse-transcription product for more reliable downstream processing.
In gene expression studies, contaminating reverse-transcription components can reduce the consistency of amplification and quantitative measurements. Removing those materials produces a cleaner cDNA template, which helps improve amplification efficiency, measurement accuracy, and reproducibility. These outcomes are especially relevant when purified material is used for PCR, quantitative PCR, sequencing, or transcriptome library preparation.