BIOC221 - Lab Lectures

What are the steps of molecular cloning?

Isolation of interest DNA
Recombination of DNA into a cloning vector
Transformation of the recombinant vector into the host cell
Replication of DNA via host cell machinery

How is DNA of interest for cloning obtained?

using PCR

What are the features within a cloning vector?

multiple cloning site
origin of replication
Selection marker
colour selection gene

What is the function of a multiple cloning site in a cloning vector?

recognisable by many different restriction enzymes allowing for vector to be cut to insert DNA of interest

What is the function of the origin of replication in a cloning vector?

recognisable by the host cell machinery that enables plasmid to be replicated

What is the function of a selection marker in a cloning vector?

gene encodes for antibiotic resistance allowing only cells which contain the vector to be selected for

What is the function of a cloning selection gene in a cloning vector?

gene which differentiates if ligation of DNA of interest into cloning vector has occured correctly, no colour = good

How does a restriction enzyme work?

using the endonuclease activity breaks phosphodiester bonds in DNA of specific palindromic sequences

How are expression vectors different from cloning vectors?

contain additionally transcription promoter, termination sequence, translation initiation sequence, and stop codon to enable transcription and translation

What are DNA libraries and why are they useful?

a collection of DNA fragments cloned into vectors
useful for identification of clone fragments and study of gene structure and function