Transcript Slide 1

Restriction Mapping
Restriction Mapping:
Double Digests
Factor I – Single Cut
Factor 2 – Single Cut
Restriction Mapping:
Double Digests
Restriction Mapping:
Double Digests
Restriction Mapping:
Double Digests
Single
BamHI
EcoRI
HindIII
PstI
5600
5600
1600
4000
5600
BamHI
EcoRI
HindIII
400
5200
600
1000
4000
600
1000
4000
PstI
5600
400
5200
600
1000
4000
ScaI
XbaI
XhoI
1800
3800
100
900
1500
3100
2000
3600
2200
3400
500
900
1100
3100
2400
3200
1200
1600
2800
500
900
1100
2600
1400
1600
2600
1800
3800
100
900
1500
3100
2000
3600
ScaI
5600
900
1400
1600
1700
XbaI
900
1600
3100
900
1200
1600
1900
XhoI
5600
Uses of Restriction Mapping
• Fingerprinting
• Restriction Fragment Length
Polymorphism Mapping
Cloning
pUC19
pUC19 Multiple Cloning Site
Ligation
AGCT
PLASMID
AGCT
KAN
TCGA
TCGA
Ligation
DNA ligase
AGCT
PLASMID
AGCT
TCGA
KAN
TCGA
Ligation
AGCT
PLASMID
AGCT
TCGA
KAN
TCGA
Ligation
AGCT
PLASMID
AGCT
TCGA
KAN
TCGA
Ligation
pUC19 digest
(50 ug/ul)
Reaction 1
Reaction 2
Reaction 3
2 ul
2 ul
2 ul
RDK1984 digest
(70 ug/ul)
6 ul
10X ligase buffer
1 ul
1 ul
water
7 ul
6 ul
T4 DNA ligase
1 ul
1 ul
1 ul