Southern Blot Protocol

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)
~undefined*DO_NOT_LET_NITROCELLULOSE_DRY_OUundefined*~Kbr_~H~M~110_Place_a_Plexiglas_plate_over_a_glass_dish_and_pour_soln_2_into_the_dish~Kbr_~H~M~111_Cut_a_piece_of_filter_paper_large_enough_to_lay_across_the_Plexiglas_and_have_the_ends_soaking_in_the_buffer~Kbr_~H~M~112_Soak_filter_paper_in_the_buffer_and_then_lay_across_the_Plexiglas~E_center_first~E_so_that_there~Kbr_~H~M~1are_NO_BUBBLES_~undefinedUse_a_plastic_pipette_to_roll_out_bubbleundefined~B~Kbr_~H~M~113_Place_gel_INVERTED_onto_filter_paper_~F~726gt~J_NO_BUBBLES~Kbr_~H~M~114_Remove_nitrocellulose_paper_from_buffer_and_lay_on_to_the_gel_~F~726gt~J_NO_BUBBLES~Kbr_~H~M~115_Soak_a_piece_of_blotting_paper_in_buffer_and_lay_it_on_top_~F~726gt~J_NO_BUBBLES~Kbr_~H~M~116_Place_a_second~E_dry~E_piece_of_blotting_paper_on_top~Kbr_~H~M~117_Place_saran_wrap_on_all_sides_of_the_gel_so_that_all_soln_must_pass_through_the_membrane_and_not_travel_around_it~Kbr_~H~M~118_Carefully_place_a_stack_of_brown_paper_towels_3~F4_inches_high_on_top_of_the_blotting_paper~Kbr_~H~M~119_Place_a_piece_of_Plexiglas_on_top_of_the_paper_towels~Kbr_~H~M~120_Center_a_full_1~F2_liter_bottle_on_top_of_the_stack~Kbr_~H~M~121_Blot_overnight~K~Hp~M~1~1~1~Kp~M~Kstrong~MDay_3~K~Hstrong~M~1~Kbr_~H~M~11_Carefully_remove_the_stack_of_paper_towels_and_blotting_paper~Kbr_~H~M~12_Remove_the_nitrocellulose~E_flip_it_over_and_write_an_L_on_the_upper_left_corner_with_a_ball_point_pen_~Afor_orientation~B~Kbr_~H~M~13_Place_it_on_filter_paper~E_view_it_under_UV_light~E_and_mark_the_lanes~Kbr_~H~M~14_Tape_lightly_b~Hw_2_pieces_of_filter_paper~Kbr_~H~M~15_Bake_@_80_degrees_in_vacuum_oven_for_2_hours~Kbr_~H~M~16_Soak_in_dd_H2O_1~F2_min~Kbr_~H~M~17_Prepare_Prehybridization_and_Hybridization_soln_and_warm_at_42_degrees~Kbr_~H~M~1~Aadd_55ul_10~7_SDS_to_11ml_Prehybridization_soln~B~Kbr_~H~M~1~Aadd_55ul_10~7_SDS_to_11ml_Hybridization_soln~B~Kbr_~H~M~18_Place_nitrocellulose_into_hybridization_tube_~ADNA_side_up~E_not_facing_glass~B~E_smooth_paper_to_walls_to_ensure_that_it_rotates_with_the_tube_and_does_not_come_free_of_the_wall~Kbr_~H~M~19_Add_prehybridization_soln~E_put_tube_into_the_hybridization_oven_for_1_� 2 hours at 42 degrees ~undefinedPrepare_probe_during_this_timundefined~B~Kbr_~H~M~110_Denature_the_probe_~F~726gt~J_add_1ul_of_1M_NaOH_for_every_9_ul_of_probe_and_put_into_37_degree_H2O_bath_for_10_minutes~Kbr_~H~M~111_Pour_out_the_prehydridization_soln~Kbr_~H~M~112_Mix_the_probe_into_the_hybridization_soln_in_the_bottom_of_the_hybridization_tube_and_then_mix_over_the_paper~Kbr_~H~M~113_Hybridize_overnight_at_42_degrees~K~Hp~M~1~1~1~Kp~M~Kstrong~MDay_4~K~Hstrong~M~1~Kbr_~H~M~11_warm_2_containers_of_Low_Stringency_buffer_to_65_degrees_in_shaker~Kbr_~H~M~12_remove_blots_and_dump_hyb_soln~Hprobe_into_proper_radioactive_waste_container~Kbr_~H~M~13_soak_blot_in_one_container_of_LS_buffer_for_15_minutes~Kbr_~H~M~14_dump_and_repeat_wash_in_LS_buffer_2_more_times~Kbr_~H~M~15_do_a_forth_wash_in_HS_buffer_as_needed.~Kbr_~H~M~16_Once_reading_is_roughly_.02_~F_.04_using_the_Geiger_counter_on_the_lowest_setting~E_place_blot~Kbr_~H~M~1between_filter_paper_to_remove_excess_buffer~Kbr_~H~M~17_tape_blot_to_filter_paper~Kbr_~H~M~18_wrap_blot_and_paper_completely_in_saran_wrap_and_tape_sealed_at_bottom_or_on_back~Kbr_~H~M~19_put_blot_and_film_in_cartridge_in_dark_room~Kbr_~H~M~110_expose_2_hours_in_�80 freezer and develop film
11 expose overnight as needed