cdna libraries and vectors

LIBRARIES

Random collection of DNA from a particular organism that contains at least one copy of every DNA segment from that being
Isolate DNA samples to analyze them:
BEFORE ANALYZING:
You need to amplify & purify the fragments
You need to make lots of copies of the segments to analyze them
Do these using vectors (?)

Two different types of libraries

CDNA library: made up of transcribed mRNA fragments
Genomic library: collections of DNA fragments that represent the genome of an organism

what are vectors

Used to amplify DNA/serve as "vehicle" for DNA.
They multiply independently of the host (bacterial) genome

Where are vectors found

Found in bacteria (and some archaea & yeast)

what is our vector

pTriplEX2

What are the important regions of the vector

PUC, Ampr, lacZ, MCS, the insert, sFilA & sFilB

what is the PUC

origin of replication (ORI) - Controls host range and copy number

what is included in the Lacz gene

Multiple cloning site (MCS), Our insert, Restriction sites (sFilA & sFilB)

how does the vector work?

gene is cut so it fits with the vector (plasmid), restriction enzyme recognizes restriction sites, DNA ligase attaches to the plasmid, insert vector into bacteria (E.coli) to amplify

significance of the ampr gene

Some bacteria can survive the presence of ampicillin because they have plasmids with the ampr gene. We let the bacterial colonies grow on the Agar plates (contain ampicillin).
If they don't grow they don't contain the plasmid.
Tells us if the plasmid is in the bacteria, not if the insert is in the plasmid

significance of the lacz gene

LacZ gene encodes the beta-galactosidase enzyme. Beta-galactosidase cleaves enzyme substrate X-gal which produces a blue colony. Our insert disrupts lacz activity, therefore, if our insert IS in the plasmid, the colony should be white.