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 Is Metacaspase carboxykinase a single or multicopy gene in E. huxleyi

4. Today’s Laboratory Objectives 1. 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 2. 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 Southern Hybridization 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 Southern Hybridization 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 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 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 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