Making nonradioactive probes
This presentation is the property of its rightful owner.
Sponsored Links
1 / 12

Making Nonradioactive Probes: PowerPoint PPT Presentation


  • 51 Views
  • Uploaded on
  • Presentation posted in: General

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.

Download Presentation

Making Nonradioactive Probes:

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Making nonradioactive probes

Making Nonradioactive Probes:

PCR DIG Labelling


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


Making nonradioactive probes

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


Making nonradioactive probes

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


  • Login