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Southern Blotting
Southern Blotting
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, A260/A280 = 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 109counts/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 EtBr 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 < 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: Nitrocellulose: Bake at 80° C for 2 hours under vacuum, between layers of
3MM paper. Nylon: Cross-link DNA to the filter with UV irradiation (120mj/cm2)
Note: At this stage, filters may be stored dry in the dark for later probing.
HYBRIDIZATION
1. Probe purification
2. Prehybridize in hybridization solution (see below) for 1 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:
5X Denhardt's Solution
100mg/ml Salmon or Herring Sperm DNA
0.1% SDS
5XSSPE
50% formamide
Filter sterilize and store at -20°C.
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):
1. TmDNA:RNA = 79.8°C + 18.5 (log10[Na]) + 0.58 (%GC) + 11.8 (%GC)2 - 0.50 (%F) (820/length)
2. Tm decreases by approximately 1° C for every 1% increase in mismatches.
3. Tm decreases by 0.5° C for every increase of 1% in formamide (%F).
4. A good hybridization temperature to begin with is 20°C below the calculated Tm.
5. 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 1x 20 min in 1X SSC, 0.1% SDS at 45° C, followed by 3 x 20 min 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.
NEXT TOPIC: Alkaline Blotting
Products Related to this Discussion:
SSC Buffer (20X)
Concentrated solution of sodium chloride-sodium citrate in distilled/deionized water.
AquaPor LE GTAC
High quality, general purpose agarose. Low boil-over. Low EEO. DNase and RNase free.
SDS Solution (20%)
Eliminates the discomfort of working with powdered SDS.
Denaturation Solution
Ready-to-use buffer solution of sodium chloride and sodium hydroxide designed for Southern and
Northern Blotting.
Neutralization Solution
Ready-to-use sodium chloride-Tris buffer solution designed for Southern and Northern Blotting.
Dextran Sulfate - ULTRA PURE
Aids in formation of networks during hybridization, thus expediting the annealing process.
Formamide - ULTRA PURE
Deionized and packed under nitrogen. Ready-to-use. Electrophoresis grade.