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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 ...
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.
The target vector is linearized and cut with a blunt-end restriction enzyme. This vector is then tailed with dideoxythymidine triphosphate (ddTTP) using terminal transferase. It is important to use ddTTP to ensure the addition of only one T residue. This tailing leaves the vector with a single 3'-overhanging thymine residue on each blunt end. [1]
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 ...
pGreenII 0000: minimal T-DNA with Left and Right border, lacZ gene for blue/white selection during cloning multiple cloning site derived from pBluescript. [5]pGreenII 62-SK: derived from pGreenII 0000, the LacZ blue/white cloning selection has been replaced with a 35S-MCS-CaMV cassette that allows the insertion of a gene of interest into a 35S over-expression cassette.
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 ...
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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.
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