Making nonradioactive probes
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Making Nonradioactive Probes:. PCR DIG Labelling. Broad Overall Objective. Is Myb61 a single or multicopy gene in A. thaliana. Research Plan. Isolate Genomic DNA. Digest Genomic DNA with Various Restriction Enzymes. Agarose Gel Electrophoresis and Southern Transfer. Southern Blot.

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Making Nonradioactive Probes:

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Making Nonradioactive Probes:

PCR DIG Labelling


Broad Overall Objective

Is Myb61 a single or multicopy gene in A. thaliana


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


Today’s Laboratory Objectives

  • To make a homologous gene probe to the

    Myb61 gene from A. thaliana

  • To learn how to set up and run a polymerase chain reaction (PCR)

  • Evaluate PCR products and the success of the reaction


Probes

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

  • Types: RNA, DNA, proteins

  • Signal: radioactive, fluorescent, enzymatic

  • Classification:

    Heterologous- from a different organism

    Homologous- 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:MYB61 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 PCR

Reaction Components:

  • Template DNA

  • Primers

  • dNTP + DIG-dUTP

  • Buffer

  • dH2O

  • Taq DNA Polymerase


How to judge the success of a PCR reaction?

Agarose gel electrophoresis is used to size PCR products.

Is a product band visible?

Are there multiple bands?

Is the band of the expected size?

Are there primer dimers?


Probe Detection

  • Blot incubated with DIG probe

  • Wash to eliminate non-specifically bound probe molecules

  • Probe detected via DIG specific antibody conjugated to alkaline phosphatase enzyme

  • Phosphatase reacts with substrate NBT/BCIP to cause a blue ppt


Next Week

  • Hybridization

  • Washes and Color Detection

  • Analysis


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