See the section Membranes Used for Southern and Northern Hybridization in Introduction: Analysis of DNA by Southern Blotting ( Green and Sambrook 2021a). Immersion in 70% alcohol for 2 min followed by drying in air. The cut off tips should be sterilized before use, either by autoclaving or by Large-bore yellow tips can be purchased or generated rapidly by cutting off the end of standard yellow tips with scissors,Ī dog nail clipper, or a sharp razor blade. Image capturing system (e.g., a CCD camera and a UV illuminator) Glass baking dishes, large enough to accommodate the gel Neutralization buffer I (for transfer to uncharged membranes) or Neutralization buffer II (for alkaline transfer to nylon membranes)Ĭross-linking device (e.g., Stratalinker, Agilent GS Gene Linker, Bio-Rad), microwave oven, or vacuum ovenĮquipment for agarose gel electrophoresis (clean, dry horizontal electrophoresis apparatus with chamber and comb) HCl (0.2 n), for depurination of DNA (optional see note to Step 6) Gel-loading buffer I or IV (6×, with sucrose) Markers radiolabeled with 35S or 33P are not recommended because they usually require exposure times that are different from those optimal for the target bands.Įlectrophoresis buffer (1× TAE or 0.5× TBE) Of the gel and a HindIII digest of bacteriophage λ DNA in the lane on the opposite side of the gel. We recommend using a 1-kb ladder (Life Technologies) in the lane closest to one side Sets of size markers are available from many commercial manufacturers, or they can be prepared by digesting cloning vectors Gel matrix may cause distortion of the DNA bands and may retard the migration of the DNA fragments to varying degrees.Īlkaline transfer buffer (for alkaline transfer to nylon membranes) or Denaturation solution (for neutral transfer), Neutral transfer buffer (either 10× SSC or 10× SSPE), and SSC (6×) Of DNA fragments may be obtained by staining the gel after electrophoresis with SYBR Gold. However, a more accurate measurement of the size

The gel may beĬast and run in the usual way in buffers containing SYBR Gold, for example. This capacity allows the entire digestion reaction to be loaded without spillage. Too little mammalian genomic DNA is transferred to allow signals from single-copy sequences to be detected in a reproducibleĪgarose gel (0.7%) cast in electrophoresis buffer (0.5× TBE or 1× TAE) in the absence of a SYBR dyeįor analysis of mammalian genomic DNA, most investigators use large gels (20 × 20 × 0.5 cm) containing 20 standard slots largeĮnough to hold ∼50–60 μL. The method therefore works best when the target sequences are present in high concentration (e.g., when analyzingĬloned DNAs or less complex genomes ). Transfer of DNAįragments is rapid, but the efficiency is low because the agarose gel quickly becomes dehydrated as fluid is withdrawn fromīoth sides. Is useful when the need arises to analyze the same set of restriction fragments with two different probes. Alternatively,ĭNA can be simultaneously transferred from the top and bottom surfaces of a single agarose gel to two membranes. The DNA is then fixed to the membrane and prepared for hybridization. The relative positions of the DNA fragments are preservedĭuring their transfer to the membrane. To a solid support (usually a nylon or nitrocellulose membrane). The DNA is then denatured in situ and transferred from the gel To size by electrophoresis through a standard agarose gel. In Southern blotting, DNA is digested with one or more restriction enzymes, and the resulting fragments are separated according

Southern blot analysis