[16] Subcloning Flashcards
What is subcloning?
The process of transferring a DNA fragment from one plasmid to another.
What are the main steps of subcloning?
- Digestion of vector and insert DNA
- Ligation of the insert into the vector
- Transformation into competent cells
- Selection of transformants
What is a restriction enzyme?
An enzyme that cuts DNA at specific recognition nucleotide sequences.
RE - Initials
Restriction Enzyme
What is the purpose of a ligation step in subcloning?
To join the DNA fragment (insert) and the vector together.
What is a DNA ligase?
An enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond.
What is transformation in the context of subcloning?
The introduction of foreign DNA into a cell.
Why is antibiotic selection used in subcloning?
To select for cells that have taken up the plasmid containing the antibiotic resistance gene.
What are competent cells in the context of subcloning?
Bacteria that have been prepared to take up foreign DNA.
What is a vector in molecular biology?
A DNA molecule used as a vehicle to transfer foreign genetic material into another cell.
Why are plasmids commonly used as vectors in subcloning?
Because they are small, circular, double-stranded DNA molecules that can replicate independently of the host cell chromosome.
What are common features of cloning vectors?
- Origin of replication
- Selectable markers
- Multiple cloning sites
What is the purpose of an origin of replication in a vector?
It is a sequence of DNA where replication begins, allowing the vector to multiply within the host.
What are selectable markers in the context of subcloning?
Genes that confer resistance to antibiotics or other toxins, used to ensure that only bacteria containing the vector will survive.
What is a multiple cloning site?
A short segment of DNA which contains many (12-20) restriction sites.
Why is a multiple cloning site useful in subcloning?
It allows for the insertion of DNA fragments at this location using various restriction enzymes.
What is a recombinant plasmid?
A plasmid that has had a particular piece of DNA inserted into it through a laboratory technique.
What is a colony in the context of subcloning?
A visible mass of microorganisms all originating from a single mother cell.
How is a colony used in subcloning?
Individual colonies are picked and grown to obtain large amounts of the cloned insert.
What is the purpose of a control in subcloning experiments?
It ensures that the observed changes are caused by the experiment and not by other factors.
