Making Nonradioactive Probes:
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Transcript of Making Nonradioactive Probes:
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Making Nonradioactive Probes:Making Nonradioactive Probes:PCR DIG LabellingPCR DIG Labelling
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Broad Overall ObjectiveBroad Overall Objective
Is Myb61 a single or multicopy Is Myb61 a single or multicopy gene in gene in A. thalianaA. thaliana
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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
Sout
hern
Blo
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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
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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 organismHomologous- from the same organismHomologous- from the same organism
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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
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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
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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
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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
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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?
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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
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Next WeekNext Week
HybridizationHybridization Washes and Color DetectionWashes and Color Detection AnalysisAnalysis