Restriction Mapping
of a Bacterial Plasmid
(Danna and Nathans, 1971)
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
Plasmids
Plasmids
Plasmids
Plasmids
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.
A Restriction Enzyme (BgII)
EcoRI
5’ G/AATTC 3’
3’ CTTAA/G 5’
CGTAGCGTAGCGAATTCGTGCGATGCAT
GCATCGCATCGCTTAAGCACGCTACGTA
EcoRI
5’ G/AATTC 3’
3’ CTTAA/G 5’
CGTAGCGTAGCG
AATTCGTGCGATGCAT
GCATCGCATCGCTTAA
GCACGCTACGTA
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
Restriction Enzyme Examples
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’
4 cutter
6 cutters
8 cutter
Restriction Map
Restriction Digest
EcoRI
4361 bp
HindIII
4361 bp
BamHI
4361 bp
AccI
ApaLI
1593 bp
2768 bp
2617 bp
1246 bp
498 bp
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
Agarose
• Agarose is very water soluble polysaccharide
• Forms porous, aqueous gels after heating
and cooling
Electrophoresis
6000
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power supply
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Gel Visualized Under UV Light
Plasmids on Agarose Gels
uncut
cut once
EXPERIMENT 1:
MAPPING DNA
• Session
agarose
• Session
#2
• Session
#3
• Session
• Session
1: single enzyme digests and
gel #1
2: double digests and agarose gel
3: more digests and agarose gel
4: run and blot gel #4
5: complete DNA blot.
Today’s Experiment
Restriction 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
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 water
1ul enzyme
Incubate for 1 hour at 37C
Add 4ul “stop mix” (50% glycerol, 1% SDS, 50mM EDTA, 0.1%
bromphenyl blue)
Restriction Enzymes for This
Experiment
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’
Your Gel Today
Size standards
XhoI
XbaI
ScaI
PstI
HindIII
EcoRI
BamHI
Size standards
Your Gel Today
Size
XhoI
XbaI
ScaI
PstI
HindIII
EcoRI
BamHI
Size
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