Making Nonradioactive Probes:

1. Making Nonradioactive Probes: PCR DIG Labelling

2. Broad Overall Objective Is Phosphoenolpyruvate carboxykinase a single or multicopy gene in E. huxleyi

3. Research Plan

4. Today�s Laboratory Objectives To make a homologous gene probe to the PEPCK gene from E. huxleyi 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

9. DIG DNA Labeling by PCR Reaction Components: Template DNA Primers dNTP + DIG-dUTP Buffer dH2O Taq DNA Polymerase

10. How does one 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. 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 emitting photons of light that can be detected via chemidoc system