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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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?