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        MicroRNA Northern Protocol

        互联网

        1151

         

        Gel Setup Using Protean II system

        Clean materials with 10% SDS and rinse thoroughly with ddH20
        Set up gel apparatus as per normal (use thick spacers) 1.5mM
        Make up 15% Denaturing Gel
        Northern Gel
        # of Gels
         
         
        40% Acrylamide
         18.75 mL
         
        5x TBE Buffer
         5 mL
         
        dH20
         10 mL
         
        Urea
         21 g
         
        10% APS
         400 ul
         
        TEMED
         40 ul
         

         Allow gel to polymerize for 1 hour
        Assemble gel apparatus and add the running buffer (0.5X TBE)
        Clean out wells with running buffer - make sure there are NO leaks!!!
        Pre-run the gel at 180 volts for 30 min
        Rinse wells right before loading sample
         

        RNA Prep and Gel Running

        You want to load 20ug of total RNA per lane - add DEPC H2O up to 20uL
        Add 20uL of formamide to your RNA sample
        Heat RNA at 65oC for 10 min
        Chill on ice for 1 min
        Add some Bromophenol Blue loading dye
        Load samples and run at 180 volts until the dye reaches the bottom of the gel
        After running gel, stain with EtBr in 0.5X TBE for 5 min. Destain in 05X TBE. This will allow you to visualize tRNAs and 5S RNA for normalization.  Place a ruler down as a reference.
        Rinse gel in 0.5X TBE to remove excess EtBr.
         

        Gel Transfer with Trans-blot Semi-Dry transfer cell

        Use the GeneScreen Plus membrane and cut to size slightly larger than the gel.
        Soak membrane in dH20 for a few seconds
        Soak membrane in transfer buffer (0.5X TBE) for 15 minutes
        Soak 2 pieces of whatman paper in 0.5X TBE
        Set up transfer as such - From Bottom (anode) to top : whatman, membrane, gel, whatman, cathode plate.  Make sure to roll out any bubbles
        Transfer at 400mAmps for 1 hour.  The voltage will start out low but increase by the end of the transfer
         

        Post Transfer

        Wash blot in 0.5X TBE to remove any traces of the gel
        Place wet membrane on a wet sheet of filter paper and UV Crosslink at optimal setting
        Store membrane at 4oC until use
         

        Labelling Probes

                       To a screw top tube, add this

                                      10.4ul dH20

                                      2ul 10x PNK Buffer

                                      2ul Oligo Probe

                                      1ul T4 PNK

                                      5ul  32P gATP

                       Mix and incubate at 37 degrees for 45 min

                       Add 80ul of TE

                       Run through G-25 column

                       Count probe

         

        Probing Membranes

        Prehyb membrane in Ultrahyb Oligo solution for 0.5 hours at 42o C.  Add 1mL/10cm2 of membrane.
        Add your 32P-labeled probe to the prehyb solution and incubate 12-24 hours at 42?C
        Pour off hyb solution and wash membrane as follows - 2 washes for 30 min. in 2xSSC/0.5% SDS
        Cover membrane in saranwrap
        Place in phosphor-imager cassette for ~4 hours.
         

        Stripping Membranes

                       Boil membrane in stripping solution (0.1X SSC, 1% SDS) for 10-30 min

         

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