plasmids circular pieces of”extrachromosomal” dna propagated inside host
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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