What I Learned about Southern Blotting By: Matthew Garma LCC A.B.E. Workshop 2006

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What I Learned aboutSouthern BlottingBy
Matthew Garma (LCC)A.B.E. Workshop 2006
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Southern Blotting

• Developed in the 1970s by Edward M. Southern
• Widely Used for DNA analysis
• Used for detecting the presence of a particular
  gene

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Southern Blotting Procedures

• Nucleic Acid Isolation
• Digest Genomic DNA and Gel Electrophoresis
• Transfer DNA to membrane
• Probe preparation
• Hybridization and DIG labeling
• Detection and Autoradiography
• Interpretation of results

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Digest Genomic DNA and perform Gel Electrophoresis

• Use restriction enzymes to cut DNA
• Load and run DNA on agarose gel (gel
  electrophoresis) with ?hindIII and 100bp ladder
  (Note molecular weight ladder should be labeled
  with DIG)

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Transfer DNA to Membrane

• Transfer DNA from gel to nitrocellulose membrane
  by capillary action

Agar gel with DNA
Weight
Wick (filter paper)
Filter paper
Paper towel stack
20X SSC Buffer
Membrane
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Probe preparation

• Use restriction enzymes to cut out the gene of
  intrest (from Plasmid DNA)
• Separate digested DNA using gel electrophoresis
• Purify gel separated DNA
• Mix purified DNA in tube with cocktail mix
  containing dNTP, random primers, DNA polymerase,
  and DIG-UTP. In this step the probe DNA is
  duplicated and labeled with DIG

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Hybridization and Detection

• Denature probe and apply to membrane for
  hybridization. Wash membrane.
• Block non-specific binding sites with Maleate
  buffer.
• Add Anti-DIG-AP antibodies to Maleate buffer
• Wash membrane and apply CSPD (in detection
  buffer) substrate to membrane

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Hybridization and Detection
Light
Anti Digoxygenin Antibodies with
Alkaline Phosphatase
CSPD
DIG-UTP
Probe (Gene of Interest)

I I I I I I I
I
DNA on membrane
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Detection and Autoradiography

• Print Southern blot from membrane onto x-ray
  film to obtain final result
• Interpret results, troubleshooting, follow up
  experiments.

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Total genomic DNA on agar gel
Southern Blot of same DNA (final result on x-ray
film)
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Group 1
MW ve WT 2A1
2A2 cut unc cut unc
cut unc
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Group 2
MW ve WT PDI 9A3
PDI9 7 unc cut un
cut un cut
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Group3
Lam Plas Wt Wt 35S 35S
2SC 2Sc un
cut un cut un cut
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Group 4
Lam pla em Wt WT 2A1 2A1 2A2 2A2
un cut un cut un
cut
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Analyzing and Concluding data

• Bands on autoradiograph indicate presence of gene
  of interest
• Consider possibilities incomplete digestion?
  Digestion of gene of interest?
• Different restriction enzymes should be used in
  future tests to obtain accurate data

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Thats it

• Mahalo