Southern Analysis:

Hybridization, Washing, and

Detection
 Research Plan
Isolate Genomic DNA

Southern Blot Analysis

Digest Genomic DNA w/ Various Restriction Enzymes

Agarose Gel Electrophoresis and Southern Transfer

Make Non-Radioactive PEPCK Probe

Hyribidize Probe to Southern Blot

Washes and Chemiluminescent Detection

Data Analysis
Broad Overall Objective

Is Phosphoenolpyruvate
carboxykinase a single or
multicopy gene in E. huxleyi
 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
 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.
 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
 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 PEPCK gene
probes

 Chemiluminescent Detection: phosphatase enzyme

conjugated to anti-DIG antibody reacts with substrate
emitting photons of light when phosphate is removed
Flow Diagram of Chemiluminescent
Detection with CSPD
Reaction Solution Time
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
 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
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
 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
 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
 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