Purification Of DNA Chapter 3 - Linköping University - Free Download PDF

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Purification of DNAChapter 3Different types of DNA Total DNA Plasmid DNA Bacteriophage DNAOften.Separate chromosomal DNA fromplasmid/phage DNA

Purification of DNAFig 3.1, s26

1 OD 0.8x109 cells/mlFig 3.2 Sid 27

Fig 3.3 Sid 28

Preparation of cellextraktFig 3.4 Sid 28

Two ways to purify DNAFig 3.5 Sid 29

Phenolextraktion, det ”old” way topurify DNAFig 3.6 Sid 30

Column ChromatographyFig 3.7 Sid 31

Precipating DNA, EthanolprecipitationRecept:1vol prov av DNA2vol 96% EtOH1/10 vol 3M NaAcetatLåt stå i -80 frysen 30 minCentrifugeraOBS svårt att se pellet!!!Fig 3.8 Sid 32

DNA purification with silicaparticles/columnFig 3.10 Sid 34

Preparation of lysateFig 3.11 Sid 35

Conformations of DNAFig 3.12 Sid 36

Alkaline denaturationFig 3.13 Sid 37

CsCl density gradient centrifugationFig 3.14 Sid 37

Detection of DNAFig 3.15 Sid 38

Purification of plasmid DNA withEtBr CsCl density gradientcentrifugationFig 3.16 Sid 38

Plasmid amplificationFig 3.17 Sid 39

Purification of phage suspensionsFig 3.18 Sid 40

Induction of lysogenic phagesFig 3.19 Sid 41

To obtain high yieldFig 3.20 Sid 41

Precipitation of phages with PEGFig 3.21 Sid 42

Preparation of ss-DNA from M13Fig 3.23 Sid 43

Manipulation of DNAChapter 4Different types of enzymes: NucleasesLigasesPolymerasesModifying enzymes

NucleaserFig 4.1 Sid 47

NucleaserFig 4.2 Sid 48

NucleaserFig 4.3 Sid 48

DNA ligase reactionFig 4.4 Sid 48

DNA polymerase reactionFig 4.5 Sid 49

Modifying enzymesFig:4.6Sid 50

Fig:4.7Sid 51

Restriction enzymesFig:4.8,Sid 52

Cleavage pattern, sticky-, bluntendsFig:4.9 Sid 54

Restrictionsmap λ-fagFig:4.10Sid 55

How to find suitable restriction sitesin a fragmentUse NEB cutter:http://tools.neb.com/NEBcutter2/

Restriction digestFig:4.11, Sid 55

ElectrophoresisFig:4.12, Sid 57

Visualisation with EtBr(alternatively SyBrsafe)Fig:4.13, 4.14, Sid 58, 59

Restrictions mapFig:4.15, Sid 60

Restriction mapFig:4.16, Sid 62

The influence of DNA size onmigration rateFig:4.17, Sid 62

Ligation, the most critical step ingene cloningFig:4.19, Sid 63

LigationFig:4.20, Sid 64

Linkers for ligationFig:4.21, Sid 65

Linkers for ligationFig:4.22, Sid 66

Example of problem with linkers!Fig:4.23, Sid 66

Fig:4.24, Sid 67

AdaptorsFig:4.25, Sid 68

Fig:4.26, Sid 68

Blunt end ligation withtopoisomerasFig:4.27, Sid 69

Blunt end ligation withtopoisomerasFig:4.28, Sid 70

Introduction of DNA into living cellsTerms: Transformation Transfection Competent cells Identification of recombinants

Production of large amount of DNAFig:5.1Sid 73

Fig:5.2, Sid 73

Transformation with competentcells (chemically modified)Fig:5.3, Sid 75

Selection of plasmidsFig:5.4, Sid 76

phenotype expressionFig:5.5, Sid 76

Insertion inactivationFig:5.6, 5.7, Sid 77

Fig:5.8, Sid 78

Cloning in pUC8 (lac Z gen)Fig:5.9, Sid 79

Insertion inactivationFig:5.10, Sid 80

In vitro packagingFig:5.11, Sid 82

Bacteriophage plaquesFig:5.12, Sid 83

Fig:5.13, Sid 84

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 3.21 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 ...