Materials needed: 0.2 N HCl DIG High labeling and detection kit 0.5 M NaOH/1.5 M NaCl 20X, 2X SSC 1.5 M NaCl/0.5 M Tris-HCl, pH 7.4 0.2M EDTA (pH 8.0) Nitrocellulose membranes Day Gel hybridization Colony Hybridization Jan. 15 Genomic DNA isolation Genomic DNA isolation (Monday) PCR amplification (TA) PCR amplification (TA) Jan. 22 Electrophoresis & transfer Ligation and transformation (Wednesday) Jan. 23 Membrane cross linking and Plates stored at 4ºC (Thursday) membrane stored at 4°C Jan. 27 - Colony lifting (Wednesday) March 17 Probe labeling and overnight Probe labeling and overnight (Monday) incubation at 37°C; probe incubation at 37°C; probe stored at -20°C (TA) stored at -20°C (TA) Prehybridization and Prehybridization and incubation for overnight incubation for overnight March 18 Adding probe and overnight Adding and overnight (Tuesday) incubation at the hybridization incubation at the hybridization temperature temperature March 19 Washes and detection Washes and detection (Wednesday) Paper towels Whatman 3MM filter paper Glass plate Sponge Pyrex dish Weight Tupperware dish/or sealable bag Shaking incubator or water bath, temperature controlled Southern Blot Lab: Detailed Protocol Day 1 Genomic DNA extraction, PCR amplification and gel electrophoresis Genomic DNA was extracted using the CTAB method and the sequence of interest was amplified using PCR. PCR set up: 2 Genomic DNA (25-100ng/l) l 2.0 l 10X buffer 0.25 l 0.25 Taq DNA polymerase l 1 Forward primer l 1 Reverse primer l Add water to 20l total volume
Day Gel hybridization Colony Hybridization Jan. 15 (Monday) Genomic DNA isolation PCR amplification (TA) Genomic DNA isolation PCR amplification (TA) Jan. 22 (Wednesday) Electrophoresis & transfer Ligation and transformation Jan. 23 (Thursday) Membrane cross linking and membrane stored at 4°C Plates stored at 4ºC Jan. 27 (Wednesday) - Colony lifting March 17 (Monday) Probe labeling and overnight incubation at 37°C; probe stored at -20°C (TA) Prehybridization and incubation for overnight Probe labeling and overnight incubation at 37°C; probe stored at -20°C (TA) Prehybridization and incubation for overnight March 18 (Tuesday) Adding probe and overnight incubation at the hybridization temperature Adding and overnight incubation at the hybridization temperature March 19 (Wednesday) Washes and detection Washes and detection Materials needed: 0.2 N HCl DIG High labeling and detection kit 0.5 M NaOH/1.5 M NaCl 20X, 2X SSC 1.5 M NaCl/0.5 M Tris-HCl, pH 7.4 0.2M EDTA (pH 8.0) Nitrocellulose membranes Paper towels Whatman 3MM filter paper Glass plate Sponge Pyrex dish Weight Tupperware dish/or sealable bag Shaking incubator or water bath, temperature controlled Southern Blot Lab: Detailed Protocol Day 1 Genomic DNA extraction, PCR amplification and gel electrophoresis Genomic DNA was extracted using the CTAB method and the sequence of interest was amplified using PCR. PCR set up: 2 l Genomic DNA (25-100ng/l) 2.0 l 10X buffer 0.25 l 0.25 l Taq DNA polymerase 1 l Forward primer 1 l Reverse primer Add water to 20l total volume