生理所黄阿敏.pptVIP

Southern, Northern and Western blotting 生理所 黃阿敏 Comparison of Southern, Northern, and Western analyses of Gene X Southern hybridization First described by E. M. Southern in 1975. Applications of Southern hybridization RFLP’s, VNTR’s and DNA fingerprinting Checking of the gene knockout mice The flow chart of Southern hybridization Southern hybridization Detection of an RFLP by Southern blotting Detection of the sickle-cell globin gene by Southern blotting Checking of the gene knockout mice Flow chart of Southern hybridization Preparing the samples and running the gel Southern transfer Probe preparation Prehybridization Hybridization Post-hybridization washing Signal detection Preparing the samples and running the gel Digest 10 pg to 10 ?g of desired DNA samples to completion. Prepare an agarose gel, load samples (remember marker), and electrophorese. Stain gel ethidium bromide solution (0.5 ?g/ml). Photograph gel (with ruler). Critical parameters (I) Note the complexity of DNA Genomic DNA A single-copy of mammalian gene, 3 Kb average in length 10 mg x 3 Kb/3 x 106 Kb = 10 mg x 1/106 = 10 pg Plasmid DNA or PCR products 0.1 mg of a 3 Kb plasmid DNA ?100 ng Gel treatment Acid treatment 0.2 N HCl solution Denaturation NaOH solution Neutralization Tris-Cl buffer (pH8.0) Southern transfer Measure gel and set up transfer assembly: Wick in tray with 20x SSC Gel Nitrocellulose or Nylon filters (soaked in H2O and 20x SSC) 3MM Whatman filter paper Paper towels Weight After Southern transfer Dissemble transfer pyramid and rinse nitrocellulose in 2x SSC Bake nitrocellulose at 80?C for 2 hr or UV-crosslink Nylon membrane for seconds Preparation of probes Synthesis of uniformly labeled double-stranded DNA probes Preparation of single-stranded probes Labeling the 5? and 3? termini of DNA Synthesis of double-stranded DNA probes Nick translation of DNA Labeled DNA probes using random oligonucleotide primers Nick translation Preparation of single-stranded pr