Southern Blotting

Southern Blotting: The Procedure


The protocols given are for Genomic Southern Blotting, one of the most sensitive Southern techniques. This set of protocols will give superior results in any standard Southern application.

SAMPLE PREPARATION

Genomic DNA samples must be extremely pure,with the A260/A280 ratio being equal to 1.8, with no nuclease contamination and minimal fragmentation.

RESTRICTION DIGESTION

Digests must be complete for results to be interpretable. It is advisable to run test reactions for the first blot, to optimize conditions and times. Incomplete digests are the primary cause of Southern Blot failure. It is particularly important to ensure that DNA has fully dissolved prior to digestion (allow 2 - 3 hours at 4° C).

A 1 kb gene fragment is present in a typical mammalian genome at 0.3 ppm. This level of representation can be detected in approximately 10 µg of digested genomic DNA, using probes (length approximately 500 bp) labeled to 109 counts/µg.

ELECTROPHORESIS (see Restriction Mapping)

Include lanes of DNA markers, as digests of genomic DNA generate a smear, not discrete bands.

  1. Use 0.7% of a low EEO agarose, run in 1X TBE.
  2. Photograph gel after ethidium bromide staining, using a fluorescent ruler to provide a reference scale.

TRANSFER

  1. Soak gel for 10 minutes in 5 volumes of 0.2N HCl. Soaking may be stopped when Bromophenol blue dye begins to turn yellow, indicating that the pH of the gel has dropped. Do not soak longer than 10 minutes.
  2. Rinse gel in 5 volumes of deionized water for 5 minutes.

    Note: This step depurinates scattered sites on the DNA. These sites are cleaved by the alkaline treatment below, enhancing the transfer of fragments > 10kb. If all fragments of interest are known to be < 10kb, this step may omitted.

  3. Soak gel for 45 minutes in denaturation solution (1.5M NaCl, 0.5M NaOH) at room temperature with gentle agitation.
  4. Rinse in deionized water 1 - 2 minutes.
  5. Soak gel for 30 minutes in neutralization solution (1M Tris pH 7.4, 1.5M NaCl) with gentle agitation.
  6. Set up capillary stack as for Northern Blotting (see protocol) using 10X SSC buffer. For recovery of small fragments less than 500 bp, use 20X SSC. Note that nylon will bind small fragments better than Nitrocellulose
  7. Allow to transfer overnight.

POST TRANSFER

  1. Disassemble the capillary stack. Mark the well positions on the membrane with pencil or indelible ink before removing it from the gel.

    Rinse the filter in 6X SSC for 5 minutes to remove any agarose fragments.

  2. Fix the DNA to the filter. For nitrocellulose, bake at 80° C for 2 hours under vacuum, between layers of 3MM paper. for nylon, cross-link DNA to the filter with UV irradiation (120mj/cm2).

HYBRIDIZATION

  1. Perform probe purification as referenced in the following page.
  2. Prehybridize in hybridization solution (see below) for one hour at 65°C, then replace with fresh solution containing probe for hybridization.

    Many different protocols exist for hybridization reactions and each must be optimized for any given probe. General guidelines are given below.

    Hybridization solution (filter sterilize and store at -20°C once mixed):

    • 5X Denhardt's Solution (see below)
    • 100mg/ml salmon or herring sperm DNA
    • 0.1% SDS
    • 5XSSPE (see below)
    • 50% formamide


    Denhardt's solution (50X):

    • 1% BSA
    • 1% Polyvinylpyrrolidone
    • 1% Ficoll


    SSPE (20X):

    • 3M NaCl
    • 0.2M Sodium Phosphate, pH 7.4
    • 25mM EDTA
       
  3. Select a hybridization temperature which will allow annealing of the probe, but prevent non-specific binding to nontarget sequences. Note that high stringency washing later in the protocol may not be able to compensate for too low a hybridization temperature. The result will be a large number of false positive bands.

    Calculation of theoretical melting temperature (Tm) is as follows

    TmDNA:RNA = 79.8°C + 18.5 (log10[Na]) + 0.58 (%GC) + 11.8 (%GC)2 - 0.50 (%F) - (820/length)

    Notes:

    • Tm decreases by approximately 1° C for every 1% increase in mismatches.
    • Tm decreases by 0.5° C for every increase of 1% in formamide (%F).
    • A good hybridization temperature to begin with is 20°C below the calculated Tm.
    • Washes should be carried out at approximately 15°C below the calculated Tm.

STRINGENCY WASHES

After hybridization, non-specifically bound probe is removed by washing in low salt buffer at high temperature. The salt concentration and temperature must be optimized for each probe/sample combination. A good starting point is to wash one time for 20 minutes in 1X SSC, 0.1% SDS at 45° C, followed by three 20 minute washes in 0.2X SSC, 0.1 % SDS at 65° C.

AUTORADIOGRAPHY

Southern Blots will need to be exposed between intensifying screens for up to 96 hours to achieve maximum sensitivity.

STRIPPING

After autoradiography, the probe may be stripped from the blot, allowing the blot to be re-probed up to 5 times (nylon) or 2 - 3 times (Nitrocellulose). Incubate the blot in 50% formamide, 6X SSC at 65°C for 30 - 60 minutes. Wrap stripped blot in plastic wrap and place on film overnight to convirm probe removal. Note: If blot is allowed to dry with probe bound to it, the probe will become permanently attached.


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