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

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

2. 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

3. 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

4. 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)

5. 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

6. 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

7. 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

8. 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

9. Detection and Autoradiography • “Print” Southern blot from membrane onto x-ray film to obtain final result • Interpret results, troubleshooting, follow up experiments.

10. Total genomic DNA on agar gel Southern Blot of same DNA (final result on x-ray film)

11. Group 1 MW +ve WT 2A1 2A2 cut unc cut unc cut unc

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

13. Group3 Lam Plas Wt Wt 35S 35S 2SC 2Sc un cut un cut un cut

14. Group 4 Lam pla em Wt WT 2A1 2A1 2A2 2A2 un cut un cut un cut

15. 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

16. That’s it • Mahalo