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Southern Blot Protocol

来源:生物谷  2007/6/25 21:40:00   访问量:6129
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Day 1
1. Digest DNA for 6 hours (or overnight)
BSA 10 mg/ml 0.5 22.65 ul of 10 ug Genomic DNA
RnaseA 10mg/ml 0.1 in TE mixed to 7.35 ul cocktail
Spermidine 100nM 0.75 per sample
10X enzyme buffer 3.0
enzyme 3.0
2. 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 2
1 mix 3ul Blue Juice per sample and load into lane
2 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, centrifuge
3 run gel at 400 – 500 mA for 3 – 4 hours or until dye has reached next comb
4 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 minutes
7 cut 2 pieces of blotting paper and 1 piece of nitrocellulose to size of the gel
8 gently place nitrocellulose on top of a layer of dd H2O and let the water soak in on its own
9 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 dish
11 Cut a piece of filter paper large enough to lay across the Plexiglas and have the ends soaking in the buffer
12 Soak filter paper in the buffer and then lay across the Plexiglas, center first, so that there
are NO BUBBLES (**Use a plastic pipette to roll out bubbles**)
13 Place gel INVERTED onto filter paper -> NO BUBBLES
14 Remove nitrocellulose paper from buffer and lay on to the gel -> NO BUBBLES
15 Soak a piece of blotting paper in buffer and lay it on top -> NO BUBBLES
16 Place a second, dry, piece of blotting paper on top
17 Place saran wrap on all sides of the gel so that all soln must pass through the membrane and not travel around it
18 Carefully place a stack of brown paper towels 3-4 inches high on top of the blotting paper
19 Place a piece of Plexiglas on top of the paper towels
20 Center a full 1-2 liter bottle on top of the stack
21 Blot overnight

Day 3
1 Carefully remove the stack of paper towels and blotting paper
2 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 lanes
4 Tape lightly b/w 2 pieces of filter paper
5 Bake @ 80 degrees in vacuum oven for 2 hours
6 Soak in dd H2O 1-2 min
7 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 wall
9 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 -> add 1ul of 1M NaOH for every 9 ul of probe and put into 37 degree H2O bath for 10 minutes
11 Pour out the prehydridization soln
12 Mix the probe into the hybridization soln in the bottom of the hybridization tube and then mix over the paper
13 Hybridize overnight at 42 degrees

Day 4
1 warm 2 containers of Low Stringency buffer to 65 degrees in shaker
2 remove blots and dump hyb soln/probe into proper radioactive waste container
3 soak blot in one container of LS buffer for 15 minutes
4 dump and repeat wash in LS buffer 2 more times
5 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 blot
between filter paper to remove excess buffer
7 tape blot to filter paper
8 wrap blot and paper completely in saran wrap and tape sealed at bottom or on back
9 put blot and film in cartridge in dark room
10 expose 2 hours in –80 freezer and develop film
11 expose overnight as needed


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