Restriction Mapping of a Bacterial Plasmid (Danna and Nathans, 1971)
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Transcript of Restriction Mapping of a Bacterial Plasmid (Danna and Nathans, 1971)
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Restriction Mappingof a Bacterial Plasmid
(Danna and Nathans, 1971)
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Plasmids
• Small, autonomously replicating extrachromosomal pieces of DNA found in bacteria, archaea and some eukaryotes
• Usually circular• Contain an origin of replication• Usually contain genes conferring advantage
on host (e.g. antibiotic resistance)• Play an important role in conjugation
(bacterial sex) and lateral gene transfer
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Plasmids
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Plasmids
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Plasmids
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Plasmids
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Restriction Enzymes
Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA at specific sequences (usually 4-8 basepairs in length)
Discovered in 1970 by Tom Kelly and Ham Smith.
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A Restriction Enzyme (BgII)
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EcoRI 5’ G/AATTC 3’3’ CTTAA/G 5’
AATTCGTGCGATGCAT GCACGCTACGTACGTAGCGTAGCGGCATCGCATCGCTTAA
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EcoRI 5’ G/AATTC 3’3’ CTTAA/G 5’
AATTCGTGCGATGCAT GCACGCTACGTA
CGTAGCGTAGCGGCATCGCATCGCTTAA
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Restriction Enzymes
• > 3,500 different restriction enzymes• > 270 different specificities
• Named for species and strain from which they were originally isolated:
– Escherichia coli R EcoRI– Bacillus amyloliquefaciens H BamHI– Providencia stuartii PstI
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MseI 5’ A/T A A 3’ 3’ T A T/A 5’
BamHI 5’ G/G A T C C 3’3’ C C T A G/G 5’
EcoRI 5’ G/A A T T C 3’3’ C T T A A/G 5’
HindIII 5’ A/A G C T T 3’ 3’ T T C G A/A 5’
NotI 5’ G C/G G C C G C 3’ 3’ C G C C G G/C G 5’
Restriction Enzyme Examples
4 cutter
6 cutters
8 cutter
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Restriction Map
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Restriction Digest
EcoRI 4361 bp
HindIII 4361 bp
BamHI 4361 bp
AccI 1593 bp 2768 bp
ApaLI 2617 bp 1246 bp
498 bp
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Agarose Gels
• To visualize the results of a restriction digest, you need to separate the different fragments of DNA, and determine their size
• We will do this by agarose gel electophoresis
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Agarose
• Agarose is very water soluble polysaccharide• Forms porous, aqueous gels after heating
and cooling
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Electrophoresis
power supply
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Gel Visualized Under UV Light
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Plasmids on Agarose Gels
uncut cut once
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EXPERIMENT 1: MAPPING DNA
• Session 1: single enzyme digests and agarose gel #1
• Session 2: double digests and agarose gel #2
• Session 3: more digests and agarose gel #3
• Session 4: run and blot gel #4• Session 5: complete DNA blot.
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Today’s ExperimentRestriction Digest of Plasmid
Each lab pair you will be given a 300µl aliquot of plasmid DNA at a concentration of approximately 100µg/ml in:TE:10mM Tris-HCl, 1mM EDTA pH 8
NOTE: This is a stock solution, you will only use a small amount for each reaction
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Restriction Digest of Plasmid
For each restriction digest, mix:5ul DNA (@100ug/ul = 0.5 ug DNA)
3ul 5x buffer (100mM NaCl, 10mM Tris-Hcl pH 7.5, 10mM MgCl2, 50 ug/ul)
6ul sterile water1ul enzyme
Incubate for 1 hour at 37C
Add 4ul “stop mix” (50% glycerol, 1% SDS, 50mM EDTA, 0.1% bromphenyl blue)
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BamHI 5’ G/G A T C C 3’3’ C C T A G/G 5’
EcoRI 5’ G/A A T T C 3’3’ C T T A A/G 5’
HindIII 5’ A/A G C T T 3’ 3’ T T C G A/A 5’
PstI 5’ C T G C A/G 3’ 3’ G/A C G T C 5’
ScaI 5’ A G T/A C T 3’ 3’ T C A/T G A 5’
XbaI 5’ T/C T A G A 3’ 3’ A G A T C/T 5’
XhoI 5’ C/T C G A G 3’ 3’ G A G C T/C 5’
Restriction Enzymes for This Experiment
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Your Gel Today
Size standards
Bam
HI
EcoR
I
HindIII
PstI
ScaI
XbaI
XhoI
Size standards
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Your Gel Today
Size
Bam
HI
EcoR
I
HindIII
PstI
ScaI
XbaI
XhoI
Size
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