Southern Blotting Sample Clauses

Southern Blotting. Genomic DNA was extracted from tails, and 15 g of DNA was digested with the appropriate enzyme in a 30 l 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 l of H2O, 500 l of salmon sperm, and 25 l 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).
Sample 1
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