Pcr Product Ligation
Pcr Product Ligation - Related Videos
07:34
DNA Ligation Reactions
0 Views •
2023
In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors – carrier DNA molecules that will replicate target fragments in host organisms. This video provides an...
03:41
Agarose Gel Electrophoresis of DNA Amplicons Post PCR: A Method to Analyze Products of Multiplex PCR and Evaluate PCR Reaction Success
0 Views •
2025
In this video, we demonstrate the separation of bacterial PCR-amplified DNA using agarose gel electrophoresis. Agarose gel functions as a molecular sieve, enabling the negatively-charged DNA to migrate based upon their size under an applied electric field.
06:01
Optimized PCR-based Detection of Mycoplasma
0 Views •
Cited by 14 •
2011
The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.
07:55
Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
0 Views •
Cited by 9 •
2022
The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.
11:53
Cecal Ligation Puncture Procedure
0 Views •
Cited by 200 •
2011
The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.
FAQs
DNA ligase seals a properly aligned DNA junction by forming a phosphodiester bond between a 5′ phosphate and a neighboring 3′ hydroxyl group. The reaction becomes possible after compatible cohesive ends or overhangs bring the vector and amplified fragment into register. This chemical joining creates a continuous recombinant DNA molecule suitable for downstream propagation or analysis.
Compatible ends allow the insert and vector to align through complementary base pairing before ligase acts. Cohesive ends or other compatible overhangs provide the recognition and positioning needed for efficient joining, whereas mismatched ends do not create the required alignment. End design therefore influences whether the desired recombinant plasmid can form and whether its orientation can be controlled.
Added restriction sites can generate defined compatible ends and support directional cloning, helping place the amplified fragment in a selected orientation within the vector. Single-base overhangs support TA cloning, which uses a different end-compatibility strategy. These choices determine how the insert and vector associate and can influence the structure of the resulting recombinant plasmid.
A typical workflow first produces the DNA fragment by PCR, prepares compatible ends on the fragment and vector, and allows those ends to align. DNA ligase then seals the junctions, producing a recombinant plasmid. The construct can subsequently be introduced into host cells, where propagation enables recovery and sequence verification of the inserted DNA.
This approach is useful when an amplified DNA fragment must be maintained in a plasmid or examined in a defined recombinant context. Following introduction into host cells, the construct can support sequence verification, gene expression studies, mutation analysis, or larger DNA assembly. The appropriate end strategy depends on whether orientation or a particular cloning format matters.
A recovered recombinant plasmid can reveal whether the amplified fragment was incorporated into the intended DNA construct through sequence verification. Once established in host cells, it may also support analysis of mutations, examination of gene expression, or construction of larger assemblies. Thus, ligation links PCR amplification to several downstream biological investigations rather than serving only as an endpoint.