Southern Analysis:

1. Southern Analysis: Hybridization, Washing, and Detection

2. Research Plan Isolate Genomic DNA Southern Blot Analysis Digest Genomic DNA w/ Various Restriction Enzymes Agarose Gel Electrophoresis and Southern Transfer Make Non-Radioactive Metacaspase Probe Hyribidize Probe to Southern Blot Washes and Chemiluminescent Detection Data Analysis

3. Broad Overall Objective Is Metacaspase carboxykinase a single or multicopy gene in E. huxleyi

4. Today’s Laboratory Objectives • To become familiar with a Southern Hybridization, Washing and Detection Methods a. mechanics and trouble spots b. What variables can be manipulated to enhance signal • Data Analysis and Interpretation • Positive control- efficacy of probe and hybridization conditions • Negative control- stringency of hybridization • Experimental signal- identify restriction fragments harboring the PEPCK gene

5. Theoretical Basis of SouthernHybridization and Washing Prehybridization: to block portions of membrane where there is no bound DNA. This will prevent probe from binding to membrane. Hybridization: Heat denatured probe added to prehybridization solution and incubated overnight. Conditions optimized to allow probe to bind to complementary sequences on membrane.

6. Theoretical Basis of SouthernHybridization and Washing • Washing: to removes non-specifically bound probe molecules. • Variables that affect stringency of washes include: salt concentration, temperature, and SDS concentration

7. Theoretical Basis of Chemiluminescent Detection • Blocking: performed with BSA to prevent non-specific binding of antibody • Antibody Wash: antibody binds to DIG portion of DIG-dUTP incorporated during amplification of Metacaspase gene probes • Chemiluminescent Detection: phosphatase enzyme conjugated to anti-DIG antibody reacts with substrate emitting photons of light when phosphate is removed

9. Flow Diagram of Chemiluminescent Detection with CSPD ReactionSolutionTime Washing 2X SSC, 0.1% SDS 10 min Washing 0.5X SSC, 0.1% SDS 30 min Blocking 0.1 M Malate, 0.15 M NaCl,1% Blocking Reagent 30 min Antibody Blocking Reagent, 150 mU/ml Anti-Dig Ab 30 min Washing 0.1 M Malate, 0.15 M NaCl, 0.3% Tween 20 30 min Detection CSPD: 0.1 M Tris, 0.1 M NaCl (1:100) 5 min Enhance 37 C incubation 15 min Document ChemiDoc XRS

10. Detection • Blot incubated with DIG probe • Wash to eliminate non-specifically bound probe molecules • Probe detected via DIG specific antibody conjugated to alkaline phosphatase enzyme • Phosphatase reacts with substrate emitting photons of light that can be detected via chemidoc system

11. Substrate belongs to group of dioxetane phenyl phosphates • Upon dephosphorylation by alkaline phosphatase intermediate is formed whose decomposition results in emission of light • Blot incubated at 37 C for 10 minutes to initiated decomposition

12. Troubleshooting Poor signal • Probe specific activity too low • Inadequate depurination • Inadequate transfer buffer • Not enough target DNA • Transfer time too short • Inefficient transfer system • Probe concentration too low • Incomplete denaturation of probe and/or target DNA • Final wash too stringent • Hybridization time too short • Inappropriate membrane

13. Troubleshooting Spotty Background • Unincorporated nucleotides not removed from labeled probe • Particles in hybridization buffer • Agarose dried on membrane • Baking or UV crosslinking when membrane contains high salt

14. Troubleshooting High Background • Insufficient Blocking • Membrane allowing to dry out during hybridization or washing • Membranes adhered during hybridization or washing • Bubbles in hybridization bag • Walls of hybridization bag collapsed on to membrane • Not enough wash solution • Hybridization temperature too low • Labeled probe molecules are too short • Probe Concentration too high • Inadequate prehybridization • Probe not denatured • Not enough SDS in wash solution