Restriction Mapping.

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Presentation transcript:

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 ScaI XbaI XhoI 5600 400 5200 600 1000 4000 1800 3800 100 900 1500 3100 2000 3600 2200 3400 500 1100 2400 3200 1600 1200 2800 2600 1400 1700 1900

Uses of Restriction Mapping Fingerprinting Restriction Fragment Length Polymorphism Mapping

Cloning

pUC19

pUC19 Multiple Cloning Site

Ligation PLASMID TCGA AGCT KAN TCGA AGCT

Ligation DNA ligase PLASMID TCGA AGCT KAN TCGA AGCT

Ligation AGCT AGCT PLASMID KAN TCGA TCGA WHY DO THIS AT LOW CONCENTRATION?

Ligation AGCT AGCT PLASMID KAN TCGA TCGA WHY DO THIS AT LOW CONCENTRATION?

Ligation Reaction 1 Reaction 2 Reaction 3 pUC19 digest (50 ug/ul) 2 ul RDK1984 digest (70 ug/ul) 6 ul 10X ligase buffer 1 ul water 7 ul T4 DNA ligase