Transcript Southern Analysis: - California State University San Marcos

Results from Probe Synthesis
do
lk
pn rd
st sw
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?
Southern Analysis:
Hybridization, Washing, and
Detection
Broad and Long Term Objective
To determine the copy number of Myb
transcription factor genes in the genome of
the model plant Arabidopsis thaliana
Research Plan
Isolate Genomic DNA
Southern Blot
Digest Genomic DNA with Various Restriction Enzymes
Agarose Gel Electrophoresis and Southern Transfer
Make Non-Radioactive Myb Probe
Hyribidize Probe to Southern Blot
Washes and Colorimetric Detection
Data Analysis
Today’s Laboratory Objectives
1.
To become familiar with a Southern Hybridization,
Washing and Detection Methods
a.
b.
2.
Mechanics and trouble spots
What variables can be manipulated to enhance signal
Data Analysis and Interpretation
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Positive control- efficacy of probe and hybridization conditions
Negative control- stringency of hybridization
Experimental signal- identify restriction fragments harboring myb
genes
Theoretical Basis of Southern
Hybridization 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.
Stringency of Hybridization
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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 + 0.7[Na+]}) + 0.41(%[G+C]) – 500/n – 1(% mismatch)
n = length of duplex (bp)
Applicable for sequences >15 bp
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.
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.
Theoretical Basis of Colorimetric Detection

Blocking: performed with BSA
to prevent non-specific binding
of antibody
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Antibody Wash: antibody
binds to DIG portion of DIGdUTP incorporated during
amplification of Myb61 gene
probe
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Colorimetric Detection:
phosphatase enzyme
conjugated to anti-DIG
antibody reacts with substrate;
when phosphate is removed
blue/purple precipitate is
formed
Flow Diagram of Colorimetric
Detection with NBT/BCIP
Reaction
Washing
Washing
Rinse
Blocking
Solution
2X SSC, 0.1% SDS
0.5X SSC, 0.1% SDS
Time
2 x 5 min
2 x 15 min
0.1 M Tris (pH 7.5), 0.15 M NaCl 1 min
0.1 M Malate, 0.15 M NaCl,0.5%
30 min
Blocking Reagent
Antibody
Blocking Reagent, 150 mU/ml
Anti-Dig Antibody
30 min
Washing
0.1 M Malate, 0.15 M NaCl, 0.3%
Tween 20
2 x 15 min
Detection
0.1 M Tris, 0.1 M NaCl, 1/50 vol
NBT/BCIP stock solution
1-12 hr
Data Analysis*
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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
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
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