Southern Blotting Sample Clauses

Southern Blotting. Genomic DNA was extracted from tails, and 15 μg of DNA was digested with the appropriate enzyme in a 30 μl reaction at 37°C overnight. Digested genomic DNA was loaded on a 1% agarose gel in 1x TAE buffer, and run overnight at 40 volts. The next day, the gel was stained with ethidium bromide for 20 minutes and then a picture was taken with a ruler. Proper digestion was detected by a smear of DNA representing all size fragments. The gel was washed in 500 ml of 0.25 HCL for 15 minutes, and then washed in 500 ml of 0.5N NaOH/1M NaCl for 15 minutes. The gel was then neutralized by washing three times in 0.5M Tris (pH 8.0) with 3M NaCl for 30 minutes each. DNA was transferred to a nitrocellulose membrane in 20X SSC (3M NaCl/0.3M sodium citrate) overnight at RT. The membrane was cross linked by UV light, and pre-hybridized in 10- 15 ml of hyb buffer (GE healthcare, NIF939) at 65°C for 1 hour. A probe was radioactivity-labeled by using the Rediprime II kit (GE healthcare, RPN1636), and once the labeled probe was spun down, 100 μl of H2O, 500 μl of salmon sperm, and 25 μl of COT DNA was added. The probe was boiled for 10 minutes, and added to the bottle containing the membrane and hybridized at 65°C overnight. The membrane was washed in 0.1xSSC/0.1% SDS twice at RT and subsequently twice at 65°C for 15 minutes per wash. The membrane was placed on film (Kodak BioMax MsFilm 8294985) at -80°C overnight and then developed on the Konica MinoLTA developer (SRX-101A).