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Making Nonradioactive Probes: PCR DIG Labelling. Broad Overall Objective Is Myb61 a single or multicopy gene in A. thaliana Is Myb61 a single or multicopy.

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Presentation on theme: "Making Nonradioactive Probes: PCR DIG Labelling. Broad Overall Objective Is Myb61 a single or multicopy gene in A. thaliana Is Myb61 a single or multicopy."— Presentation transcript:

1 Making Nonradioactive Probes: PCR DIG Labelling

2 Broad Overall Objective Is Myb61 a single or multicopy gene in A. thaliana Is Myb61 a single or multicopy gene in A. thaliana

3 Research 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 Southern Blot

4 Today’s Laboratory Objectives  To make a homologous gene probe to the Myb61 gene from A. thaliana 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

5 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

6 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

7 PCR Template DNA: MYB61 in pENTR 221 Myb61 mRNA Template = 1.496 Kb M13 Rev GGAAACAGCTATGACCATG M13 For GTAAAACGACGGCCAGTG

8 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

9 DIG DNA Labeling by PCR Reaction Components:  Template DNA  Primers  dNTP + DIG-dUTP  Buffer  dH 2 O  Taq DNA Polymerase

10 How to judge the success of 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? Are there multiple bands? Is the band of the expected size? Are there primer dimers?

11 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

12 Next Week  Hybridization  Washes and Color Detection  Analysis


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