Southern Northern and Western blotting

Southern Northern and Western blotting - Southern, Northern...

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Southern, Northern and Western blotting o o &&
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Comparison of Southern, Northern, and Western analyses of Gene X
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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
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Southern hybridization Transfer buffer
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Detection of an RFLP by Southern blotting
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Detection of the sickle-cell globin gene by Southern blotting
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Checking of the gene knockout mice
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Flow chart of Southern hybridization Preparing the samples and running the gel Southern transfer Probe preparation Prehybridization Hybridization Post-hybridization washing Signal detection Isotope Non-isotope
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Preparing the samples and running the gel Digest 10 pg to 10 μ g of desired DNA samples to completion. Prepare an agarose gel, load samples (remember marker), and electrophorese. Stain gel ethidium bromide solution (0.5 μ g/ml). Photograph gel (with ruler).
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Critical parameters (I) Note the complexity of DNA Genomic DNA A single-copy of mammalian gene, 3 Kb average in length 10 μ g x 3 Kb/3 x 10 6 Kb = 10 μ g x 1/10 6 = 10 pg Plasmid DNA or PCR products 0.1 μ g of a 3 Kb plasmid DNA 2245 100 ng
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Gel treatment Acid treatment 0.2 N HCl solution Denaturation NaOH solution Neutralization Tris-Cl buffer (pH8.0)
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Southern transfer Measure gel and set up transfer assembly: Wick in tray with 20x SSC Gel Nitrocellulose or Nylon filters (soaked in H 2 O and 20x SSC) 3MM Whatman filter paper Paper towels Weight
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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
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This note was uploaded on 05/19/2011 for the course BIO 20 taught by Professor Churva during the Spring '11 term at Mapúa Institute of Technology.

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Southern Northern and Western blotting - Southern, Northern...

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