Southern Blot Protocol
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Day 1
1. Digest DNA for 6 hours (or overnight)
BSA 10 mg/ml 0.5 22.65 μl of 10 μg Genomic DNA
RnaseA 10mg/ml 0.1 in TE mixed to 7.35 μl 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 μl TAE, 10-20 μl 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 μl 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)
(add 55μl 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 ~undefinedPrepare_probe_during_this_timundefined~B~K~Hp~M~Kp~M~K~Hp~M~Kp~M10_Denature_the_probe_~F~8gt~J_add_1ul_of_1M_NaOH_for_every_9_μl 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 0.02 - 0.04 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
