Limiting Dilution Cloning
Limiting Dilution Cloning - Related Videos
08:01
Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish
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Cited by 35 •
2011
Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.
09:55
Molecular Cloning
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2023
Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant DNA fragments in host organisms. The DNA fragment, which may be a gene, can be isolated from a prokaryotic or eukaryotic specimen. Following isolation of the fragment of interest, or insert, both the vector and insert must be cut with restriction enzymes and purified. The purified pieces are joined together though a technique called...
01:27
Reproductive Cloning
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2019
Reproductive cloning is the process of producing a genetically identical copy—a clone—of an entire organism. While clones can be produced by splitting an early embryo—similar to what happens naturally with identical twins—cloning of adult animals is usually done by a process called somatic cell nuclear transfer (SCNT). Somatic Cell Nuclear Transfer In SCNT, an egg cell is taken from an animal and its nucleus is removed, creating an enucleated egg. Then a somatic cell—any cell that is not a sex...
05:21
Culturing and Harvesting of HSPC Clones: A Method to Obtain the Singly-Sorted HSPC Clones
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2023
This video describes the protocol for isolating HSPCs from the bone marrow and sorting them using a fluorescent activated cell sorting technique, which is a method to obtain singly-sorted HSPCs. We culture these sorted cells to obtain HSPC clones.
02:59
Solution Concentration and Dilution
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2020
The relative amount of a given solution component is known as its concentration. Often, though not always, a solution contains one component with a concentration that is significantly greater than that of all other components. This component is called the solvent and may be viewed as the medium in which the other components are dispersed or dissolved. Solutions in which water is the solvent are, of course, very common on our planet. A solution in which water is the solvent is called an aqueous...
FAQs
Poisson statistics estimate the probability that a well receives zero, one, or multiple cells when a diluted suspension is distributed across wells. Targeting an average occupancy below one cell per well increases the likelihood of isolated-cell wells, but it does not guarantee single-cell occupancy. These estimates therefore guide dilution choices and help interpret the resulting colonies.
Low density reduces the chance that multiple starting cells enter the same well and later form a mixed colony. When an isolated cell expands, its descendants can be evaluated as a candidate clone with greater genetic or phenotypic uniformity. The approach is especially useful when consistent cell behavior or biological material is required.
A colony from a low-occupancy well is not automatically proven clonal because the well may have received more than one cell. Limiting dilution lowers that probability rather than eliminating it. Follow-up verification is therefore important when researchers need to validate a stable phenotype, genetic uniformity, or a reliable source of consistent biological material.
The process begins with a cell suspension from a mixed population, followed by serial dilution to reduce the number of cells delivered to each well. The diluted suspension is then distributed across wells at low density, often aiming for an average of less than one cell per well. Wells containing growing colonies can subsequently be selected for analysis.
Researchers can select colonies that arise from isolated-cell wells and expand them as candidate cell lines. Comparing their behavior or phenotype helps identify populations with the desired stable characteristics. This workflow supports clonal cell-line development and genetic screening, while subsequent clonality checks strengthen confidence that the selected line originated from a single cell.
Limiting dilution cloning can separate individual cells from a mixed population so that selected descendants can be expanded and evaluated for consistent properties. In hybridoma research, this supports the development of clonal populations used for monoclonal antibody production. The same strategy also helps validate that the biological material comes from a population with the intended stable phenotype.