Plasmidscircular pieces of”extrachromosomal” DNA propagated inside host• have origin of replication-> ensures host will copy it

Plasmidscircular 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

Plasmidscircular 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 Enzymesenzymes which cut DNA at specific sites

Restriction Enzymesenzymes which cut DNA at specific sitescalled "restriction enzymes" because restrict host range for certain bacteriophage

Restriction Enzymesenzymes which cut DNA at specific sitescalled "restriction enzymes" because restrict host range for certain bacteriophagebacterial” immune system”: destroy any “non-self” DNA

Restriction Enzymesbacterial” immune system” destroy “non-self”DNA-> methylase recognizes same sequence in host DNA and protects it by methylating it

Restriction Enzymesenzymes which cut DNA at specific sitesUseful for fingerprinting: each cuts a particular DNA into a distinctive set of fragments

Restriction Enzymesenzymes which cut DNA at specific sitesUseful for fingerprinting: each cuts a particular DNA into a distinctive set of fragments

Restriction Enzymesenzymes which cut DNA at specific sitesUseful for fingerprinting: each cuts a particular DNA into a distinctive set of fragmentscan map each site by double digests

Restriction Enzymes

Restriction enzymes create unpaired "sticky ends” which anneal with any complementary sequence

Restriction EnzymesRestriction enzymescreate unpaired "sticky ends” which anneal with any complementary sequencemake “recombinant DNA” by mixing 2 different fragments digested with same enzyme and gluing them together with ligase

Restriction EnzymesRestriction enzymescreate unpaired "sticky ends” which anneal with any complementary sequencemake “recombinant DNA” by mixing 2 different fragments digested with same enzyme and gluing them together with ligaseGenetic Engineering

Restriction mappingBand distance

traveledsize

1 4 21226

2 5.5 "5000"

3 5.7 4268

4 6 3530

5 7.5 2027

6 7.7 1904

7 8.4 1709

8 9 1375

9 10.5 947

10 11 831

11 11.8 564

Restriction mappingBand distance

traveledsize

1 4 21226

2 5.5 "5000"

3 5.7 4268

4 6 3530

5 7.5 2027

6 7.7 1904

7 8.4 1709

8 9 1375

9 10.5 947

10 11 831

11 11.8 564

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 fingerprintingUse DNA sequences that vary between individuals

DNA fingerprintingUse DNA sequences that vary between individuals

DNA fingerprintingUse DNA sequences that vary between individualsMicrosatellitesshort sequences 2-5 bases long repeated multiple times

DNA fingerprintingMicrosatellitesshort sequences 2-5 bases long repeated multiple times

vary between families but not within families

DNA fingerprintingSTRs (simple tandem repeats)

vary between families but not within familiesDetect by PCR with primers that bind outside repeat