Plasmids circular pieces of”extrachromosomal” DNA propagated inside host have origin of replication -> ensures host will copy it
Plasmids circular pieces of”extrachromosomal” DNA propagated inside host have origin of replication -> ensures host will copy it Have selectable marker (usually a drug-resistance gene) -> ensures cell won’t “lose” it
Plasmids circular pieces of”extrachromosomal” DNA propagated inside host have origin of replication -> ensures host will copy it Have selectable marker (usually a drug-resistance gene) -> ensures cell won’t “lose” it pGLO also carries gene for Jellyfish Green Fluorescent Protein
Restriction Enzymes enzymes which cut DNA at specific sites
Restriction Enzymes enzymes which cut DNA at specific sites called "restriction enzymes" because restrict host range for certain bacteriophage
Restriction Enzymes enzymes which cut DNA at specific sites called "restriction enzymes" because restrict host range for certain bacteriophage bacterial” immune system”: destroy any “non-self” DNA
Restriction Enzymes bacterial” immune system” destroy “non-self”DNA -> methylase recognizes same sequence in host DNA and protects it by methylating it
Restriction Enzymes enzymes which cut DNA at specific sites Useful for fingerprinting: each cuts a particular DNA into a distinctive set of fragments
Restriction Enzymes enzymes which cut DNA at specific sites Useful for fingerprinting: each cuts a particular DNA into a distinctive set of fragments
Restriction Enzymes enzymes which cut DNA at specific sites Useful for fingerprinting: each cuts a particular DNA into a distinctive set of fragments can map each site by double digests
Restriction Enzymes Restriction enzymes create unpaired "sticky ends” which anneal with any complementary sequence
Restriction Enzymes Restriction enzymes create unpaired "sticky ends” which anneal with any complementary sequence make “recombinant DNA” by mixing 2 different fragments digested with same enzyme and gluing them together with ligase
Restriction Enzymes Restriction enzymes create unpaired "sticky ends” which anneal with any complementary sequence make “recombinant DNA” by mixing 2 different fragments digested with same enzyme and gluing them together with ligase Genetic Engineering
Restriction mapping
Restriction mapping
Transformation Physical transfer of new DNA into cell 1. Preincubation: chills cells, allows cations to neutralize negative charges on plasma membrane.
Transformation 1. Preincubation: chills cells, allows cations to neutralize negative charges on plasma membrane. 2. Incubation: allows DNA to diffuse through cell wall to contact plasma membrane.
Transformation 2. Incubation: allows DNA to diffuse through cell wall to contact plasma membrane. 3. Heat shock: opens holes in plasma membrane, allows plasmid to enter by diffusion.
Transformation 3. Heat shock: opens holes in plasma membrane, allows plasmid to enter by diffusion. 4. Recovery: cells must make proteins encoded by new DNA molecule before they can be plated.
Transformation 5. Selecting transformants: cells which have taken up the plasmid are only ones which can grow in the presence of ampicillin. Form colony at place they landed on plate
DNA fingerprinting Use DNA sequences that vary between individuals
DNA fingerprinting Use DNA sequences that vary between individuals
DNA fingerprinting Use DNA sequences that vary between individuals Microsatellites short sequences 2-5 bases long repeated multiple times
DNA fingerprinting Microsatellites short sequences 2-5 bases long repeated multiple times vary between families but not within families
DNA fingerprinting STRs (simple tandem repeats) vary between families but not within families Detect by PCR with primers that bind outside repeat