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.
PCR DIG Labelling
Is Myb61 a single or multicopy gene in A. thaliana
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
Myb61 gene from A. thaliana
Heterologous- from a different organism
Homologous- from the same organism
Myb61 mRNA Template = 1.496 Kb
M13 Rev GGAAACAGCTATGACCATG
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
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?