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CMB2000 Lecture 1
Isolating DNA
Learning outcomes
CMB2000 Lecture 2
1. Denature 94C
GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
CTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
CMB2000 Lecture 2
Hybridisation
3. Extension
GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
CTCTCCCTTCAG
TTGACAGT
CTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
CMB2000 Lecture 2
GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
CTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
CMB2000 Lecture 2
GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
ACCG
AGGG
CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
CMB2000 Lecture 2
GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
ACCG
AGGG
CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
CMB2000 Lecture 2
GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
ACCG
AGGG
CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
CMB2000 Lecture 2
1. Denature 94C
2. Anneal primers
3. Extension
GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
ACCG ACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
GGATGGCTGCTATTTCCAAAACTGTCAGAGAGGG
CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
CMB2000 Lecture 2
1. Denature 94C
2. Anneal primers
3. Extension
GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
ACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
GATGGCTGCTATTTCCAAAACTGTCAGAGAGGG
CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
CMB2000 Lecture 2
2
4
1
0
1. Denature 94C
2. Anneal primers
3. Extension
GATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC
ACCG ACGATAAAGGTTTTGACAGTCTCTCCC
TGGCTGCTATTTCCAAAACTGTCAGAG AGGG
ACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
GATGGCTGCTATTTCCAAAACTGTCAGAGAGGG
ACCG ACGATAAAGGTTTTGACAGTCTCTCCC
TGGCTGCTATTTCCAAAACTGTCAGAGAGGG
CCTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG
CMB2000 Lecture 2
1. Denature 94C
2. Anneal primers
3. Extension
1
1
CMB2000 Lecture 2
3
8
1. Denature 94C
2. Anneal primers
3. Extension
1
2
CMB2000 Lecture 2
4
16
1. Denature 94C
2. Anneal primers
3. Extension
1
3
CMB2000 Lecture 2
10
1024
30
,
, ,
1073741824
1. Denature 94C
2. Anneal primers
3. Extension
1
4
CMB2000 Lecture 2
1
5
CMB2000 Lecture 2
Log
Insufficient reagents to complete extension
DNA polymerase makes mistakes
Must be ultra-sterile in your lab skills
Problem sequences
1
6
CMB2000 Lecture 2
1
7
Diagnostics
Agricultural
management
Forensics
PCR
Phylogeny
Basic
research
Drug
development
CMB2000 Lecture 2
1
8
CMB2000 Lecture 2
1
9
CMB2000 Lecture 2
94C
52C
72C
2
0
CMB2000 Lecture 2
94C
52C
Thermocyclers
72C
2
1
CMB2000 Lecture 2
2
2
CMB2000 Lecture 2
2
3
CMB2000 Lecture 2
2
4
CMB2000 Lecture 2
2
5
Primers
Synthetic oligonucleotides 18-30 bases long
Length only as long as required for specificity
Annealing temperature must be similar for
each primer in the pair
Ideally have a G or a C at the 3-end
Avoid a T because most common to mismatch
5-CAGTCAACTGCTAC-3 good, but
5-CAGTCAACTGCTGC-3 would be even better
CMB2000 Lecture 2
GAATTC
CTTAAG
2
6
CMB2000 Lecture 2
Isolating DNA
Learning outcomes
Describe genetic transformation
techniques including the use of plasmids
and vectors
2
7
2
8
CMB2000 Lecture 2
GAATTC
CTTAAG
GAATTC
CTTAAG
Cut with EcoRI
G
AATTC
CTTAA
G
G
AATTC
CTTAA
G
DNA ligase
(+ ATP)
G AATTC
CTTAAG
Recombinant DNA molecule
2
9
CMB2000 Lecture 2
AATTC
G
G
CTTAA
DNA to insert
Plasmid vector
EcoRI
G TAA
CT
AATT
C
G
DNA ligase
ATP
Recombinant plasmid
3
0
Transformation
CMB2000 Lecture 2
Transformation
heat shock
1. Cold CaCl2
CMB2000 Lecture 2
Transformation
heat shock
1. 42C quickly
CMB2000 Lecture 2
3
3
CMB2000 Lecture 2
gen
e
pIC19
H
2.7 kb
Origin
of Replication
3
4
CMB2000 Lecture 2
pIC19
H
2.7 kb
Ampr
Origin
of Replication
Plate on
ampicillin
3
5
3
6
CMB2000 Lecture 2
Gen
e
X-gal
pIC19
H
2.7 kb
CH2OH
O
HO
O
-galactosidase
-peptide. Inactive
Br
Cl
OH
Truncated -galactosidase
(lacks -peptide). Inactive
pUC
H
N
OH
HO
Non-covalent association
of two inactive components
giving active -galactosidase
CH2OH
O
OH
OH
H
N
Br
HO
OH
Cl
Further reaction to
blue insoluble dye
CMB2000 Lecture 2
Gen
e
pIC19
H
2.7 kb
Lac Z
gene
Origin
of Replication
Multiple Cloning
Site
(polylinker)
3
7
3
8
CMB2000 Lecture 2
Blue colonies
Amp
Apr r
Ap
Gen plasmid
pIC19H
size
e approx
2700bp
pUC18
LacZ
LacZ
2.7 kb
Lac
Z
Ap
Ap
gene
r
pblt101 recombinant
Origin
size
approx
3200bp
of Replication
LacZ
EcoRI
restriction
enzyme sites in
MCS
White colonies
3
9
CMB2000 Lecture 2
Gen
e
pIC19H plasmid
size approx
2700bp
Ampr
Blue colonies
Apr r
Ap
Plate on
ampicillin
plus X-gal
pUC18
LacZ
LacZ
2.7 kb
EcoRI
White colonies
Lac
Z
Ap
Ap
gene
r
pblt101 recombinant
restriction
enzyme sites in
MCS
Origin
size
approx
All
colonies
are Ampr
of3200bp
Replication
But only some are recombinant
(white ones)
LacZ
Blue/white selection of
recombinant E. coli clones
CMB2000 Lecture 2