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Making Nonradioactive Probes:

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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  1. Making Nonradioactive Probes: PCR DIG Labelling

  2. Broad Overall Objective 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 Southern Blot Make Non-Radioactive Myb Probe Hyribidize Probe to Southern Blot Washes and Colorimetric Detection Data Analysis

  4. 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

  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 • dH2O • Taq DNA Polymerase

  10. 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?

  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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