1. What I Learned about Southern Blotting By: 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 20X SSC Buffer Wick (filter paper) Filter paper Agar gel with DNA Membrane Paper towel stack Weight

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 DNA on membrane Anti Digoxygenin Antibodies with Alkaline Phosphatase DIG-UTP Probe (Gene of Interest) I I I I CSPD Light

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