Plasmid Sequencing
Plasmid Sequencing - Related Videos
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.
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.
01:28
Plasmids
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2025
Plasmids are extrachromosomal DNA molecules found in bacteria, archaea, and some eukaryotic microbes like yeast. These small, circular DNA structures typically contain fewer than 30 genes, although some may exist linearly. Plasmids vary in their number within a cell, known as copy number. Single-copy plasmids are present in one copy per cell and multi-copy plasmids are present in multiple copies, reaching over 100 copies per cell.Plasmids usually replicate independently of the chromosomal DNA...
07:37
CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion
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2017
Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.
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.
FAQs
Plasmid sequencing compares sequence reads with an assembled sequence or an expected reference. This comparison can reveal whether the insert is present in the intended form and whether the surrounding plasmid structure contains unexpected changes. By examining the full construct rather than only a selected region, researchers can identify mutations, insertions, deletions, or rearrangements that may otherwise remain unnoticed.
These changes can alter the genetic content or organization of an engineered plasmid, even when the overall construct appears correct. A sequence-level analysis identifies such differences and helps researchers determine whether the plasmid still matches its intended design. Detecting these changes before downstream work is important because they could affect transfection, protein expression, or functional experimental results.
Assembly combines generated sequence reads into a representation of the plasmid, while reference alignment compares that representation with an expected sequence. Together, these approaches support structural verification and help localize differences within the construct. The resulting comparison can show whether genes, regulatory elements, and cloned inserts are present as intended or contain sequence changes requiring further attention.
A typical workflow begins with isolation of purified plasmid DNA. The DNA is then used to generate sequence reads, which are assembled or aligned to a reference sequence for interpretation. Researchers review the resulting sequence information for the expected insert, genes, regulatory elements, mutations, insertions, deletions, and rearrangements before deciding whether the construct is suitable for subsequent experiments.
Researchers can perform plasmid sequencing as a quality-control step after constructing or isolating a clone and before using it in transfection, protein expression, or functional experiments. This timing allows the construct to be checked while corrections are still possible. It also helps confirm that an engineered variant has the intended sequence before experimental results are attributed to its biological design.
Sequence verification connects the physical plasmid used in an experiment with its intended genetic design. In biology, this supports reliable studies of cloned constructs, engineered variants, gene content, and regulatory elements. Identifying sequence discrepancies before downstream testing helps researchers interpret experimental outcomes more confidently and distinguish effects of the planned construct from effects caused by unintended genetic changes.