mbb 331 lec 3

genes are cloned by insertion into cloning vectors process

cleaving the vector with RE’S

cleaving the eukaryotic chromosome with RE’S

ligating( wiht dna ligas) the eukaryote pieces to vector

introducing the new vector to the host cell

dna isolation

lyse cells ( detergent, soap)

remove debris ( centrifuge)

separate DNA from proteins( CsCl gradient centrifugation)

concentrate by ethanol precipitation

RE’s terminology

sticky end generators provide overhands

Blunt end generators provide blunt ends

plasmids

plasmids are a circular piece of ddsna that replicates seperatly from host dna

plasmids are naturally occurring but often the ones used for gene cloning are engineered

plasmids have features that allow them to survive and grow in eukaryotic cells

typicall features:

ORI

plasmid selection markers

recognition sequences for re’s

small in size

lacZ is disrupted by cloning dna into vector ( produces white instead of blue)

polylinker?

it is the region added in the plasmids that has sequeces which are recognized and cleaved by sequences

transformation methods

chemical or electroporation

genomic library

• set of clones containing dna fragments derived directly from the genome of a single organism

a collection of dna fragments made from cutting up a dna into a lot of pieces

the fragements are then added to vectors which are stored in cells

cDNA library

• a library that includes only dna sequences trasncribed from mrna

• mrna is reverse transcribed from rna

• requires:

• - polytailed mrna

• - oligoprimer reverse trancriptase

rna/dna probes are used for ?

they are used for detecting specific dna fragments

they must be single stranded dna probe and complementary

process

make labeled prob

expose probe to dna

isolated label dna

to make a radioactive probe we use T4 polynucleotide kinase

screening for gene of interest

plaque hybridization for DNA - DNA

Southern Blots for DNA 0 DNA

Northern blots for DNA RNA( measures rna gene expresssion