Making Nonradioactive Probes:Making Nonradioactive Probes:

PCR DIG LabellingPCR DIG Labelling

Broad Overall ObjectiveBroad Overall Objective

Is Myb61 a single or multicopy Is Myb61 a single or multicopy gene in gene in A. thalianaA. thaliana

Research PlanResearch Plan

Isolate 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

So

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Today’s Laboratory ObjectivesToday’s Laboratory Objectives

To make a homologous gene probe to theTo make a homologous gene probe to the

Myb61 gene from Myb61 gene from A. thalianaA. thaliana To learn how to set up and run a polymerase chain To learn how to set up and run a polymerase chain

reaction (PCR)reaction (PCR) Evaluate PCR products and the success of the reactionEvaluate PCR products and the success of the reaction

ProbesProbes

Definition: signal molecule that is used to identify a nucleic Definition: signal molecule that is used to identify a nucleic acid or protein of interestacid or protein of interest

Types: RNA, DNA, proteinsTypes: RNA, DNA, proteins Signal: radioactive, fluorescent, enzymaticSignal: radioactive, fluorescent, enzymatic Classification: Classification:

Heterologous- from a different organismHeterologous- from a different organism

Homologous- from the same organismHomologous- from the same organism

Why DIG As a Signal Molecule?

DIGoxigenin is a steroid hapten from Digitalis lanata

A system for labeling nucleic acids and proteins

Detection Options: color, fluorescence, chemiluminescence

Faster, safer, and sensitive replacement for radioactivity

PCR Template DNA:PCR Template DNA:MYB61 in pENTR 221MYB61 in pENTR 221

Myb61 mRNA Template = 1.496 Kb

M13 Rev GGAAACAGCTATGACCATG

M13 For

GTAAAACGACGGCCAGTG

Polymerase Chain Reaction

Three Major Cycling Steps

Denaturation at 95°C for 45 sec

Annealing at 52-60°C for 30 sec

Elongation at 72 C° for 2 min

DIG DNA Labeling by PCRDIG DNA Labeling by PCR

Reaction Components:Reaction Components: Template DNATemplate DNA PrimersPrimers dNTP + DIG-dUTPdNTP + DIG-dUTP BufferBuffer dHdH22OO Taq DNA PolymeraseTaq DNA Polymerase

How to judge the success of How to judge the success of a PCR reaction?a PCR reaction?

Agarose gel electrophoresis is used to size PCR products.Agarose gel electrophoresis is used to size PCR products.

Is a product band visible?Is a product band visible?

Are there multiple bands?Are there multiple bands?

Is the band of the expected size?Is the band of the expected size?

Are there primer dimers?Are there primer dimers?

Probe DetectionProbe Detection

Blot incubated with DIG probeBlot incubated with DIG probe Wash to eliminate non-specifically bound probe moleculesWash to eliminate non-specifically bound probe molecules Probe detected via DIG specific antibody conjugated to Probe detected via DIG specific antibody conjugated to

alkaline phosphatase enzymealkaline phosphatase enzyme Phosphatase reacts with substrate NBT/BCIP to cause a blue Phosphatase reacts with substrate NBT/BCIP to cause a blue

ppt ppt

Next WeekNext Week

HybridizationHybridization Washes and Color DetectionWashes and Color Detection AnalysisAnalysis