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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Broad overall objective
Broad Overall Objective

Is Myb61 a single or multicopy gene in A. thaliana


Research plan
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
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
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
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
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
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
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
Next Week

  • Hybridization

  • Washes and Color Detection

  • Analysis


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