Southern Blot Protocol

Day 11. Digest DNA for 6 hours (or overnight)BSA 10 mg/ml 0.5 22.65 ul of 10 ug Genomic DNARnaseA 10mg/ml 0.1 in TE mixed to 7.35 ul cocktailSpermidine 100nM 0.75 per sample10X enzyme buffer 3.0enzyme 3.02. Pour a large 1% gel (5 grams agarose in 500 ul TAE, 10-20 ul 10mg/ml EtBr / 500ml of gel), cover and place in fridge till ready to use

Day 21 mix 3ul Blue Juice per sample and load into lane2 add 2 ul blue juice to marker mix (Add .5ug unlabeled lambda Hind III to ~3500 counts(~.5 ul) of 32P labeled lambda, heat lambda to 56 degrees for 3 minutes, chill on ice 1 minute, centrifuge3 run gel at 400 � 500 mA for 3 � 4 hours or until dye has reached next comb4 take a picture of the gel (with ruler)5 soak gel in Soln 1 on shaker for 15 min, repeat with new soln 1 (denaturing the DNA)6 soak gel in soln 2 on shaker for 30 min, repeat with new soln for 20 minutes7 cut 2 pieces of blotting paper and 1 piece of nitrocellulose to size of the gel8 gently place nitrocellulose on top of a layer of dd H2O and let the water soak in on its own9 once completely soaked, drain H2O and let paper soak in transfer buffer (soln 2)***DO NOT LET NITROCELLULOSE DRY OUT***10 Place a Plexiglas plate over a glass dish and pour soln 2 into the dish11 Cut a piece of filter paper large enough to lay across the Plexiglas and have the ends soaking in the buffer12 Soak filter paper in the buffer and then lay across the Plexiglas, center first, so that thereare NO BUBBLES (**Use a plastic pipette to roll out bubbles**)13 Place gel INVERTED onto filter paper -%26gt; NO BUBBLES14 Remove nitrocellulose paper from buffer and lay on to the gel -%26gt; NO BUBBLES15 Soak a piece of blotting paper in buffer and lay it on top -%26gt; NO BUBBLES16 Place a second, dry, piece of blotting paper on top17 Place saran wrap on all sides of the gel so that all soln must pass through the membrane and not travel around it18 Carefully place a stack of brown paper towels 3-4 inches high on top of the blotting paper19 Place a piece of Plexiglas on top of the paper towels20 Center a full 1-2 liter bottle on top of the stack21 Blot overnight

Day 31 Carefully remove the stack of paper towels and blotting paper2 Remove the nitrocellulose, flip it over and write an L on the upper left corner with a ball point pen (for orientation)3 Place it on filter paper, view it under UV light, and mark the lanes4 Tape lightly b/w 2 pieces of filter paper5 Bake @ 80 degrees in vacuum oven for 2 hours6 Soak in dd H2O 1-2 min7 Prepare Prehybridization and Hybridization soln and warm at 42 degrees(add 55ul 10% SDS to 11ml Prehybridization soln)(add 55ul 10% SDS to 11ml Hybridization soln)8 Place nitrocellulose into hybridization tube (DNA side up, not facing glass), smooth paper to walls to ensure that it rotates with the tube and does not come free of the wall9 Add prehybridization soln, put tube into the hybridization oven for 1 � 2 hours at 42 degrees (**Prepare probe during this time**)10 Denature the probe -%26gt; add 1ul of 1M NaOH for every 9 ul of probe and put into 37 degree H2O bath for 10 minutes11 Pour out the prehydridization soln12 Mix the probe into the hybridization soln in the bottom of the hybridization tube and then mix over the paper13 Hybridize overnight at 42 degrees

Day 41 warm 2 containers of Low Stringency buffer to 65 degrees in shaker2 remove blots and dump hyb soln/probe into proper radioactive waste container3 soak blot in one container of LS buffer for 15 minutes4 dump and repeat wash in LS buffer 2 more times5 do a forth wash in HS buffer as needed.6 Once reading is roughly .02 - .04 using the Geiger counter on the lowest setting, place blotbetween filter paper to remove excess buffer7 tape blot to filter paper8 wrap blot and paper completely in saran wrap and tape sealed at bottom or on back9 put blot and film in cartridge in dark room10 expose 2 hours in �80 freezer and develop film11 expose overnight as needed