Southern Analysis:

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Southern Analysis

• Hybridization, Washing, and Detection

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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
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Broad Overall Objective

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

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Todays 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

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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.
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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

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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

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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 (1100)
  5 min
• Enhance 37 C incubation 15 min
• Document ChemiDoc XRS

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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

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• 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

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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

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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

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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