Plasmid Amplification
Plasmid Amplification - Related Videos
02:41
Amplification of a Suicide Plasmid in a Bacterial Host
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2025
Source: Tomás, J. M., et al. Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility. J. Vis. Exp. (2022).This video demonstrates the initiator-dependent amplification of a suicide plasmid in E. coli. The plasmid, which carries an antibiotic resistance gene, replicates only in the presence of an initiator protein encoded by the host. After electroporation and incubation in recovery medium, the mixture is plated on antibiotic-containing agar.
07:11
Homemade Site Directed Mutagenesis of Whole Plasmids
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Cited by 91 •
2009
Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.
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.
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.,...
10:12
Protein Misfolding Cyclic Amplification of Prions
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Cited by 7 •
2012
Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.
FAQs
The plasmid origin is the site that directs host replication machinery to duplicate the circular DNA molecule. As bacterial cells divide, this origin-guided copying allows plasmid molecules to persist and increase throughout the culture. Because replication depends on the host, successful amplification requires both entry into suitable cells and continued maintenance during growth.
Competent cells can receive plasmid DNA during transformation, establishing the starting population for amplification. Without successful DNA entry, subsequent culture growth and selection cannot enrich plasmid-bearing bacteria. This step therefore connects purified input DNA with biological replication, while the host cell supplies the machinery needed to copy the plasmid as the population expands.
Antibiotic selection enriches the bacterial population for cells carrying the plasmid. Cells that acquire and maintain the selected plasmid are favored during growth, whereas cells lacking it are not enriched under the selection condition. This makes the culture more suitable for recovering plasmid DNA rather than relying on transformation alone.
Recovery proceeds by harvesting the culture, breaking open the bacterial cells, removing cellular contaminants, and recovering the plasmid DNA. These stages separate the target circular DNA from the biological material that supported its production. The resulting preparation provides the DNA needed for subsequent molecular biology experiments.
Researchers amplify plasmids when they need sufficient DNA for cloning, gene expression, sequencing, or mutagenesis studies. The same approach also supplies plasmid material for recombinant protein investigations. Its value lies in connecting a small amount of starting DNA with enough recovered material to support multiple downstream experimental objectives.
Amplified plasmids provide recoverable DNA that can be used in experiments examining gene expression or recombinant protein production. They also support sequence analysis and intentional DNA modification through mutagenesis. In this way, amplification serves as an enabling step that supplies the molecular construct required to investigate how introduced genetic information functions.