Results from Probe Synthesis

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Results from Probe Synthesis
pn st
rd sw
do lk
Wed, Thurs lab results
Escobar/Read
DIG
-DIG

• increased extension time (2 min, extension rate
  of Taq 50-100 nt/min, Taq
• Tgo polymerase mix extension rate ?)
• reagent contamination?

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Southern Analysis

• Hybridization, Washing, and Detection

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Broad and Long Term Objective
To determine the copy number of Myb transcription
factor genes in the genome of the model plant
Arabidopsis thaliana
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Research Plan
Isolate Genomic DNA
Digest Genomic DNA with Various Restriction
Enzymes
Agarose Gel Electrophoresis and Southern Transfer
Southern Blot
Make Non-Radioactive Myb Probe
Hyribidize Probe to Southern Blot
Washes and Colorimetric Detection
Data Analysis
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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 myb genes

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Theoretical Basis of SouthernHybridization and
Washing
Prehybridization prehybridization solution
contains a mix of proteins and nucleic acids that
will bind to the membrane, covering regions where
there is no fixed DNA (membrane blocking). This
prevents the single stranded probe from binding
nonspecifically to the membrane. Hybridization
Heat denatured probe is then added to the
prehybridization solution and incubated
overnight. On a fully blocked membrane, probe can
associate with the membrane only by hybridizing
with complementary ssDNA sequences fixed to the
membrane. Washing removes probe molecules that
are weakly associated with the surface of the
membrane or the genomic DNA.
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Stringency of Hybridization

• The incubation conditions during the
  hybridization and washing steps can be varied to
  require greater or lesser complementarity between
  probe and bound DNA (stringency)
• Stringency is determined primarily by salt
  concentration, temperature, and the
  presence/absence of organic solvents (esp.
  formamide)
• Successful hybridization between probe and a
  target DNA is determined by the Tm
• Tm (ºC) 81.5 16.6 log10 (Na/1.0
  0.7Na) 0.41(GC) 500/n 1( mismatch)
• n length of duplex (bp)
• Applicable for sequences gt15 bp

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DIG Detection Principle

• DIG labeled probes that hybridized to a target
  sequence are detected with an anti-DIG antibody
  that is covalently attached to a phosphatase
  enzyme.
• If the blot is incubated with suitable reagents
  like NBT and BCIP, phosphatase activity is
  detected by a color reaction.

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Color Development

• Substrate BCIP and NBT form a redox system
• BCIP is oxidized by the alkaline phosphatase to
  indigo by release of a phosphate group
• NBT is reduced to diformazan
• Reaction products form a water insoluble dark
  blue to brownish precipitate, depending on the
  type of membrane.

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Theoretical Basis of Colorimetric 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
  Myb61 gene probe
• Colorimetric Detection phosphatase enzyme
  conjugated to anti-DIG antibody reacts with
  substrate when phosphate is removed blue/purple
  precipitate is formed

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Flow Diagram of Colorimetric Detection with
NBT/BCIP

• Reaction Solution Time
• Washing 2X SSC, 0.1 SDS 2 x 5 min
• Washing 0.5X SSC, 0.1 SDS 2 x 15 min
• Rinse 0.1 M Tris (pH 7.5), 0.15 M NaCl 1 min
• Blocking 0.1 M Malate, 0.15 M NaCl,0.5 30 min
• Blocking Reagent
• Antibody Blocking Reagent, 150 mU/ml 30 min
• Anti-Dig Antibody
• Washing 0.1 M Malate, 0.15 M NaCl, 0.3 2 x 15
  min
• Tween 20
• Detection 0.1 M Tris, 0.1 M NaCl, 1/50 vol 1-12
  hr
• NBT/BCIP stock solution

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Data Analysis

• What information do your positive and negative
  controls provide?
• How many hybridizing fragments for each
  restriction enzyme- what does this indicate about
  Myb gene copy number?
• How homologous is Myb61 to other gene sequences?
  (BLASTn) From your blot, does it appear that
  these sequences hybridized with the Myb61 probe?

Evidence for a single copy gene
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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

• 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