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TA cloning (also known as rapid cloning or T cloning) is a subcloning technique that avoids the use of restriction enzymes [1] and is easier and quicker than traditional subcloning. The technique relies on the ability of adenine (A) and thymine (T) (complementary basepairs) on different DNA fragments to hybridize and, in the presence of ligase ...
Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3'-end of the PCR products. This characteristic is exploited in "sticky end" TOPO TA cloning. [1] For "blunt end" TOPO cloning, the recipient vector does not have overhangs and blunt-ended DNA fragments can be cloned.
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. [1] The cloning vector may be DNA taken from a virus , the cell of a higher organism, or it may be the plasmid of a bacterium.
A multiple cloning site is located within lacZ-α, and an insert successfully ligated into the vector will disrupt the gene sequence, resulting in an inactive β-galactosidase. Cells containing vector with an insert may be identified using blue/white selection by growing cells in media containing an analogue of galactose . Cells expressing β ...
Molecular cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the same in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replication, and the resulting recombinant DNA is introduced into the organism from which the replication sequences were obtained ...
This property may be exploited in TA cloning where the ends of the PCR product can anneal to the T end of a vector. TA ligation is therefore a form of sticky end ligation. Blunt-ended vectors may be turned into vector for TA ligation with dideoxythymidine triphosphate (ddTTP) using terminal transferase.
In recombinant DNA technology, the ccdB gene is widely used as a positive selection marker (e.g. the Invitrogen's Zero Background and Gateway cloning vectors). [7] In August 2016, the CcdB positive selection technology falls completely within the public domain and is now fully free for personal or commercial use.
MAP-Seq; Maxam–Gilbert sequencing; Methods to investigate protein–protein interactions; Microbial dark matter; Microsatellite enrichment; Minusheet perfusion culture system; MNase-seq; Molecular cloning; Multi-parametric surface plasmon resonance; Mutagenesis (molecular biology technique)
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