Purification of DNA
Chapter 3
Different types of DNA
• Total DNA
• Plasmid DNA
• Bacteriophage DNA
Often..
Separate chromosomal DNA from
plasmid/phage DNA
Purification of DNA
Fig 3.1, s26
1 OD=0.8x109 cells/ml
Fig 3.2 Sid 27
Fig 3.3 Sid 28
Preparation of cellextrakt
Fig 3.4 Sid 28
Two ways to purify DNA
Fig 3.5 Sid 29
Phenolextraktion, det ”old” way to
purify DNA
Fig 3.6 Sid 30
Column Chromatography
Fig 3.7 Sid 31
Precipating DNA, Ethanol
precipitation
Recept:
1vol prov av DNA
2vol 96% EtOH
1/10 vol 3M NaAcetat
Låt stå i -80 frysen 30 min
Centrifugera
OBS svårt att se pellet!!!
Fig 3.8 Sid 32
DNA purification with silica
particles/column
Fig 3.10 Sid 34
Preparation of lysate
Fig 3.11 Sid 35
Conformations of DNA
Fig 3.12 Sid 36
Alkaline denaturation
Fig 3.13 Sid 37
CsCl density gradient centrifugation
Fig 3.14 Sid 37
Detection of DNA
Fig 3.15 Sid 38
Purification of plasmid DNA with
EtBr CsCl density gradient
centrifugation
Fig 3.16 Sid 38
Plasmid amplification
Fig 3.17 Sid 39
Purification of phage suspensions
Fig 3.18 Sid 40
Induction of lysogenic phages
Fig 3.19 Sid 41
To obtain high yield
Fig 3.20 Sid 41
Precipitation of phages with PEG
Fig 3.21 Sid 42
Preparation of ss-DNA from M13
Fig 3.23 Sid 43
Manipulation of DNA
Chapter 4
Different types of enzymes:
•
•
•
•
Nucleases
Ligases
Polymerases
Modifying enzymes
Nucleaser
Fig 4.1 Sid 47
Nucleaser
Fig 4.2 Sid 48
Nucleaser
Fig 4.3 Sid 48
DNA ligase reaction
Fig 4.4 Sid 48
DNA polymerase reaction
Fig 4.5 Sid 49
Modifying enzymes
Fig:4.6Sid 50
Fig:4.7Sid 51
Restriction enzymes
Fig:4.8,Sid 52
Cleavage pattern, sticky-, bluntends
Fig:4.9 Sid 54
Restrictionsmap λ-fag
Fig:4.10Sid 55
How to find suitable restriction sites
in a fragment
Use NEB cutter:
http://tools.neb.com/NEBcutter2/
Restriction digest
Fig:4.11, Sid 55
Electrophoresis
Fig:4.12, Sid 57
Visualisation with EtBr
(alternatively SyBrsafe)
Fig:4.13, 4.14, Sid 58, 59
Restrictions map
Fig:4.15, Sid 60
Restriction map
Fig:4.16, Sid 62
The influence of DNA size on
migration rate
Fig:4.17, Sid 62
Ligation, the most critical step in
gene cloning
Fig:4.19, Sid 63
Ligation
Fig:4.20, Sid 64
Linkers for ligation
Fig:4.21, Sid 65
Linkers for ligation
Fig:4.22, Sid 66
Example of problem with linkers!
Fig:4.23, Sid 66
Fig:4.24, Sid 67
Adaptors
Fig:4.25, Sid 68
Fig:4.26, Sid 68
Blunt end ligation with
topoisomeras
Fig:4.27, Sid 69
Blunt end ligation with
topoisomeras
Fig:4.28, Sid 70
Introduction of DNA into living cells
Terms:
• Transformation
• Transfection
• Competent cells
• Identification of recombinants
Production of large amount of DNA
Fig:5.1Sid 73
Fig:5.2, Sid 73
Transformation with competent
cells (chemically modified)
Fig:5.3, Sid 75
Selection of plasmids
Fig:5.4, Sid 76
phenotype expression
Fig:5.5, Sid 76
Insertion inactivation
Fig:5.6, 5.7, Sid 77
Fig:5.8, Sid 78
Cloning in pUC8 (lac Z gen)
Fig:5.9, Sid 79
Insertion inactivation
Fig:5.10, Sid 80
In vitro packaging
Fig:5.11, Sid 82
Bacteriophage plaques
Fig:5.12, Sid 83
Fig:5.13, Sid 84
