Colony Pcr Screening
Colony Pcr Screening - Related Videos
08:51
Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)
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Cited by 9 •
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Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.
02:41
Clonal Isolation and PCR Screening of GFP-Excised Recombinant Herpesvirus Retaining the Target Gene
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Source: Tang, N., et al. Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing. J. Vis. Exp. (2019)This video demonstrates the clonal isolation and PCR-based screening of recombinant herpesvirus following site-specific excision of a GFP marker. Fluorescence-activated cell sorting is used to isolate non-fluorescent infected cells, and junction PCR confirms retention of the target gene in GFP-negative clones.
06:44
Keratinocyte Colony Formation Assay: An In Vitro Assay to Assess the Ability of Keratinocytes to Grow into Colonies
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In this video, we perform a clonogenic assay of keratinocytes harvested from adult mice to determine the ability of individual cells to proliferate and form colonies. This keratinocyte harvesting technique is useful for studying other downstream aspects of cutaneous chemical carcinogenesis and tumor promotion.
08:50
Genetic Screens
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Genetic screens are critical tools for defining gene function and understanding gene interactions. Screens typically involve mutating genes and then assessing the affected organisms for phenotypes of interest. The process can be “forward”, where mutations are generated randomly to identify unknown genes responsible for the phenotypes, or it can be “reverse”, where specific genes are targeted for mutation to observe what phenotypes are produced.Here, JoVE reviews various types of genetic...
07:47
ScanLag: High-throughput Quantification of Colony Growth and Lag Time
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Cited by 66 •
2014
ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.
FAQs
Primer placement determines what the screen can reveal. Primers flanking an insert test whether amplification spans the expected inserted region, whereas primers spanning a junction test whether two DNA elements are connected at the intended boundary. This distinction lets researchers assess either insert presence and approximate size or a specific assembly junction, depending on the cloning question.
Direct use of colony material makes the screen efficient because cells supply the PCR template without first requiring plasmid isolation. That shortcut allows many transformed colonies to be compared early in the workflow and limits reagent use. Positive candidates can then proceed to plasmid isolation and sequencing, which provide follow-up characterization beyond the initial screen.
Gel electrophoresis converts the amplification reaction into a size-based readout. A product at the expected approximate size supports the presence of a candidate construct when the selected primers target the relevant insert or junction. Because the readout is based on amplification and product size, the screen serves as an initial verification step before more detailed analysis.
A practical workflow begins by taking a small amount of cells from each transformed colony and using that material directly as the PCR template. Researchers choose primers for the insert or its junction, run the amplification, and examine the products by gel electrophoresis. Colonies showing the expected result can be advanced to plasmid isolation and sequencing.
During recombinant DNA assembly, the method helps compare candidate colonies for the presence of the intended insert or assembly junction before researchers invest in plasmid preparation. This early check narrows the set of clones requiring follow-up work. Its value is greatest within workflows that need rapid selection of candidates for downstream confirmation or experiments.
In strain engineering and library construction, many colonies may represent different candidate clones. Testing colony material directly provides an efficient way to identify colonies carrying the desired construct before further characterization. The resulting shortlist can support downstream experiments by focusing plasmid isolation, sequencing, or other analyses on clones that already show the expected PCR outcome.