Molecular biology protocols – DNA – RFLP

Data analysis for fingerprinting and diversity study

Large-Scale DNA Extraction 1
DNA Extraction Using the Sap Extractor 5
Small-Scale Extraction of High Quality DNA 7
Quantification and Quality Control of DNA 11
Molecular Weight Markers for Gel Electrophoresis 13
Neutral Agarose Gel Electrophoresis 18
Double-Thick Gels 19
RFLP Flow Chart 20
Restriction Digests of Genomic DNA 21
Southern Blotting onto Non-Charged Membranes 24
PCR Amplification of Inserts from Plasmids 26
PCR Amplification of Inserts from Bacterial Cultures 27
Incorporation of Digoxigenin-dUTP into Plasmid Inserts Using PCR 28
Relative Quantification of Amplified Inserts in Gel 30
Checking the Activity of Incorporated Digoxigenin-dUTP 32
Hybridization and Detection of Dig-Labeled Probes 33
Removal of Probe for Re-Use of Membranes 37
STS and SSR Protocols 38
DNA Fingerprinting of Maize and Wheat Using an Automatic DNA Sequencer 48
Chemiluminescent AFLP protocol 53
Detecting Transgenic DNA Sequences in Maize 60
Plasmid Mini-Preps 67
Isolation of Plasmid Inserts 69
Preparation of Frozen Competent Cells 71
Bacterial Transformations 72
General Stock Solutions 73
Beckmann DU-65 Spectrophotometer DNA Quantification Program 77
Data Sheets 80
Notes 81


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