Lecture13 Restriction Enzymes

Front 1

endonuclease

Back 1

make cuts within a DNA chain

Front 2

exonuclease

Back 2

digests DNA from the ends

Front 3

restriction sites

Back 3

short inverted repeat sequences

Front 4

restriction digestion

Back 4

generates a reproducible set of DNA fragments

Front 5

3 regions of plasmid DNA

Back 5

ORI- sequence of 50-100 nucleotides that allows for plasmid DNA replication

drug resistance gene- allows for selection of recombinant plasmid

region of foreign DNA insertion- where you put foreign DNA

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plasmid dna

Back 6

-circular double stranded dna (separate from bacterias chromosme)

-parasitic or aparasitic
-replicates independently from chromosomal host DNA bc differnet origins of replication
- around 3kb
- occurs naturally in bacteria, yeast, and lower eukaryotic systems

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transformation

Back 7

introduction of plasmid into the cell
2 methods:
1) treatment of cells with Cesium Chloride (punches holes in membrane)
2)electroporation - use of electric pulse results in transient changes in the membrane

1) mix recombinant plasmid with ecoli cells w/ cacl2; heat pulse
2) culture on nutrient agar plate w/ ampicillin
3)transformed cell survives or cells that do not take up plasmid die on ampicillin plates

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ampicillin

Back 8

inhibits cell wall synthesis (antibiotic)

Front 9

tetracyclin

Back 9

inhibits protein synthesis
binds to 30s ribosomal subunit
interferes with the binding of tRNA to mRNA

Front 10

chloramphenicol

Back 10

inhibits protein synthesis by binding to 70s ribosome subunit

Front 11

polylinker region

Back 11

artifical dna region that contains many restriction sites

foreign dna is inserted here

Front 12

restriction enzymes

Back 12

bacterial enzymes that recognize specific 4- 8 base pair sequences in double stranded DNA

cleave both dna strands at restriction sites

cut the DNA into a reproducible set of fragments (restriction fragments)

Front 13

bacteriophage lambda

Back 13

~49 kbp DNA
high transformation efficiency
can insert large bp sequences, up to 25 bp foreign dna

lambda virions can be assembled in vitro

Front 14

lambda virion

Back 14

infectious virus particle consists of head and tail

head contains viral DNA genome
tail serves to infect E.coli cells