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        NORTHERN BLOT PROTOCOL1

        丁香园论坛

        1970
        NORTHERN BLOT OF RNA GEL

        Buffers:

        20XSSPE (500 ml):
        3.6M NaCl : 105.2 g
        0.2M phosphate buffer pH 7.0: 100mL of 1M
        20mM EDTA: 20mL of 0.5M

        Phosphate buffer pH7.0 (500ml):
        NaH2PO4 (monobasic) 140 mL of 1M
        Na2H2PO4 (Dibasic) 360 mL of 1M
        pH to 7.0 after both are added

        Hybridization buffer =HB (100mls):
        5X SSPE: 25mls 20X
        2% SDS: 20 mls 10%
        ~undefinedRight_before_using_add~Kbr_~H~M~2~11000ug_denatured_RNAse_free_CT_DNA_~Aheat_to_95o_for_3_min_to_denature~B~Kbr_~H~M~2~15000ug_yeast_RNA~Kbr_~H~M~2~1~Kbr_~H~M~2~1WASH~I~Kbr_~H~M~2~12X_SSPE_~D_0.1~7_SDS_~A500_mls~B~Kbr_~H~M~2~150_mls_20X~Kbr_~H~M~2~15_mls_10~7_SDS~L_0.1~7_SDS~Kbr_~H~M~2~1~Kbr_~H~M~2~1Running_buffer_~A1L~B~I~Kbr_~H~M~2~1100_mls_10X_MOPS~Kbr_~H~M~2~152.6_mls_of_formaldehyde~Kbr_~H~M~2~1847.4_mls_H20~Kbr_~H~M~2~1~Kbr_~H~M~2~1RNA_Gel_~A70ml~B~I~Kbr_~H~M~2~10.84_g_agarose~Kbr_~H~M~2~17_mls_10x_MOPS~Kbr_~H~M~2~158.38_mls_H2O~Kbr_~H~M~2~1put_gel_in_solution_by_heating~Kbr_~H~M~2~1let_cool_to_60°C
        add Formaldehyde to equal 0.66M --3.78 mls
        pour gel, in hood if possible

        Run gel
        after, wash gel with distilled water
        put on posiblotter

        layers from top to bottom:

        sponge
        3-4 Whatman paper cut to size
        ( both of these up above should be soaked in 10X SSPE but not dripping)
        gel
        mask --make sure the gel overlays the mask so that it can seal
        membrane with line of the top of gel and the right top corner marked
        3 pieces slightly bigger 3M paper
        hard plastic with holes
        clamp on and make sure there is a good seal, pressure 75-80 mm Hg for1 hr or more
        blot membrane dry, cut top right corner
        wrap in saran wrap and UV irradiate, RNA side down for 2.5 min
        wash couple min in hot 2X SSPE/0.1% SDS sol'n (microwave 30-45sec, 50% power)
        air dry

        Pre Hybridize, 6 hrs-overnight:
        set oven to 65°C
        heat HB buffer to put SDS in solution
        roll up membrane inside piece of mesh and put into hybridizaton tube(2XSSPE/0.1%SDS)
        check for air bubbles
        put tube in oven balanced with another tube on opposite side

        Labeling probe High Prime (BMC):
        1) put 25ng of probe in a total volume of 11uL with ddH2O
        2) denature @ 95°C 3 min.
        3) quick cool on wet ice
        4) add 4ul High Prime (Boehringer Mannheim)
            5ul alpha P32 dCTP 3000uCi/mmole
            20ul total
        5) incubate 10-15 min 37°C
        6) add 28uL of 1X TE, 2ul 0.5M EDTA, to 20ul reaction
        7) prespin G25 column for 1 minute
        8) Add 50ul sample to column and spin for 2.5 min in clinical centrifuge
        9) throw away column
        10) mix in a new tube 100ug CT-DNA
            50ug Yeast RNA
             in a 0.5mL tube
        12) denature 95° 3 min
        13) add to hybrid tube mix, hybridize at 65°C for 20-36 hrs

        Post hybridization:
        hybridize for 36-48 hrs
        wash
        1) heat up 2X SSPE/0.1% SDS (wash) heat up to 65° C (?use microwave?to warm)
        2) take out tube
        3) pour liquid iinto hot radioactive waste
        4) rinse with 10ml wash do this twice and pour waste out
        5) put in 40 to 50 mls of wash and shake vigoursly
        6) put in oven for 20 min rotating
        7) pour down sink
        8) another 40-50mls and shake, oven
        9) pour down sink and make sure it is no longer hot and then take out membrane
        10) wash on shaker with 0.2XSSPE w/ 0.1% SDS at RT for 15 min.
        11) air dry, expose yourself
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