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 gene in A. thaliana

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

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

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 Myb61 mRNA Template = 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 Reaction Components:  Template DNA  Primers  dNTP + DIG-dUTP  Buffer  dH 2 O  Taq DNA Polymerase

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?

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  Hybridization  Washes and Color Detection  Analysis