Results from Probe Synthesis

Results from Probe Synthesis Results from Probe Synthesis pnst

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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:Southern Analysis:

Hybridization, Washing, and Hybridization, Washing, and DetectionDetection

Broad and Long Term ObjectiveBroad and Long Term Objective

To determine the copy number of MybTo determine the copy number of Myb

transcription factor genes in the genome oftranscription factor genes in the genome of

the model plant the model plant Arabidopsis thalianaArabidopsis thaliana

Research PlanResearch PlanIsolate Genomic DNA

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

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Today’s Laboratory ObjectivesToday’s Laboratory Objectives

1.1. To become familiar with a Southern Hybridization, To become familiar with a Southern Hybridization, Washing and Detection MethodsWashing and Detection Methodsa. Mechanics and trouble spotsa. Mechanics and trouble spotsb. What variables can be manipulated to enhance signal b. What variables can be manipulated to enhance signal

2.2. Data Analysis and InterpretationData Analysis and Interpretation Positive control- efficacy of probe and hybridization conditionsPositive control- efficacy of probe and hybridization conditions Negative control- stringency of hybridizationNegative control- stringency of hybridization Experimental signal- identify restriction fragments harboring myb Experimental signal- identify restriction fragments harboring myb

genes genes

Theoretical Basis of SouthernTheoretical Basis of SouthernHybridization and WashingHybridization 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 HybridizationStringency 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 + 0.7[Na+]}) + 0.41(%[G+C]) – 500/n – 1(% mismatch)

n = length of duplex (bp)

Applicable for sequences >15 bp

DIG Detection PrincipleDIG Detection Principle

DIG labeled probes that hybridized to a target sequence DIG labeled probes that hybridized to a target sequence are detected with an anti-DIG antibody that is covalently are detected with an anti-DIG antibody that is covalently attached to a phosphatase enzyme. attached to a phosphatase enzyme.

If the blot is incubated with suitable reagents like NBT and If the blot is incubated with suitable reagents like NBT and BCIP, phosphatase activity is detected by a color reaction.BCIP, phosphatase activity is detected by a color reaction.

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.

Color DevelopmentColor Development

Theoretical Basis of Colorimetric DetectionTheoretical Basis of Colorimetric Detection

Blocking: performed with BSA to Blocking: performed with BSA to prevent non-specific binding of prevent non-specific binding of antibodyantibody

Antibody Wash: antibody binds Antibody Wash: antibody binds to DIG portion of DIG-dUTP to DIG portion of DIG-dUTP incorporated during amplification incorporated during amplification of Myb61 gene probeof Myb61 gene probe

Colorimetric Detection: Colorimetric Detection: phosphatase enzyme phosphatase enzyme conjugated to anti-DIG antibody conjugated to anti-DIG antibody reacts with substrate; when reacts with substrate; when phosphate is removed phosphate is removed blue/purple precipitate is formedblue/purple precipitate is formed

Flow Diagram of Colorimetric Flow Diagram of Colorimetric Detection with NBT/BCIPDetection with NBT/BCIP

ReactionReaction SolutionSolution TimeTimeWashingWashing 2X SSC, 0.1% SDS2X SSC, 0.1% SDS 2 x 5 min2 x 5 minWashingWashing 0.5X SSC, 0.1% SDS0.5X SSC, 0.1% SDS 2 x 15 min2 x 15 minRinseRinse 0.1 M Tris (pH 7.5), 0.15 M NaCl0.1 M Tris (pH 7.5), 0.15 M NaCl 1 min1 minBlockingBlocking 0.1 M Malate, 0.15 M NaCl,0.5%0.1 M Malate, 0.15 M NaCl,0.5% 30 min30 min

Blocking ReagentBlocking Reagent

AntibodyAntibody Blocking Reagent, 150 mU/ml Blocking Reagent, 150 mU/ml 30 min30 minAnti-Dig AntibodyAnti-Dig Antibody

WashingWashing 0.1 M Malate, 0.15 M NaCl, 0.3%0.1 M Malate, 0.15 M NaCl, 0.3% 2 x 15 min2 x 15 minTween 20Tween 20

DetectionDetection 0.1 M Tris, 0.1 M NaCl, 1/50 vol0.1 M Tris, 0.1 M NaCl, 1/50 vol 1-12 hr1-12 hrNBT/BCIP stock solutionNBT/BCIP stock solution

Data AnalysisData Analysis** What information do your positive and negative controls provide?What information do your positive and negative controls provide? How many hybridizing fragments for each restriction enzyme- what How many hybridizing fragments for each restriction enzyme- what

does this indicate about Myb gene copy number?does this indicate about Myb gene copy number? How homologous is Myb61 to other gene sequences? (BLASTn) How homologous is Myb61 to other gene sequences? (BLASTn)

From your blot, does it appear that these sequences hybridized with From your blot, does it appear that these sequences hybridized with the Myb61 probe?the Myb61 probe?

Evidence for a single copy gene

TroubleshootingTroubleshooting

Poor signalPoor signal Probe specific activity too lowProbe specific activity too low Inadequate depurinationInadequate depurination Inadequate transfer bufferInadequate transfer buffer Not enough target DNANot enough target DNA Transfer time too shortTransfer time too short Inefficient transfer systemInefficient transfer system Probe concentration too lowProbe concentration too low Incomplete denaturation of probe and/or target DNAIncomplete denaturation of probe and/or target DNA Final wash too stringentFinal wash too stringent Hybridization time too shortHybridization time too short Inappropriate membraneInappropriate membrane

TroubleshootingTroubleshooting

High BackgroundHigh Background Insufficient BlockingInsufficient Blocking Membrane allowing to dry out during hybridization or washingMembrane allowing to dry out during hybridization or washing Membranes adhered during hybridization or washingMembranes adhered during hybridization or washing Bubbles in hybridization bagBubbles in hybridization bag Walls of hybridization bag collapsed on to membraneWalls of hybridization bag collapsed on to membrane Not enough wash solutionNot enough wash solution Hybridization temperature too lowHybridization temperature too low Labeled probe molecules are too shortLabeled probe molecules are too short Probe Concentration too highProbe Concentration too high Inadequate prehybridizationInadequate prehybridization Probe not denaturedProbe not denatured Not enough SDS in wash solutionNot enough SDS in wash solution