What I Learned about

Southern Blotting

By: Matthew Garma (LCC)A.B.E. Workshop 2006

Southern Blotting

• Developed in the 1970’s by Edward M. Southern

• Widely Used for DNA analysis

• Used for detecting the presence of a particular gene

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

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)

Transfer DNA to Membrane

• Transfer DNA from gel to nitrocellulose membrane by capillary action

20X SSC Buffer

Wick (filter paper) Filter paper

Agar gel with DNA

Membrane

Paper towel stack

Weight

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

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

Hybridization and Detection

DNA on membrane

Anti Digoxygenin Antibodies with

Alkaline Phosphatase

DIG-UTP

Probe (Gene of Interest)

I I I I I I I II I I I I I I I

CSPDCSPD

Light

Detection and Autoradiography

• “Print” Southern blot from membrane onto x-ray film to obtain final result

• Interpret results, troubleshooting, follow up experiments.

Total genomic DNA on agar gel

Southern Blot of same DNA (final result on x-ray film)

Group 1

MW +ve WT 2A1 2A2 cut unc cut unc cut unc

Group 2MW +ve WT PDI 9A3 PDI9 7 unc cut un cut un cut

Group3

Lam Plas Wt Wt 35S 35S 2SC 2Sc un cut un cut un cut

Group 4

Lam pla em Wt WT 2A1 2A1 2A2 2A2 un cut un cut un cut

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

That’s it

• Mahalo