Transferring

1. Fill reservoir with 10X SSPE. Lay a piece of chromatography paper across a glass
   plate. Allow the SSPE to wick up the paper (or just soak the paper wet).
2. Remove all bubbles between the paper and the plate by rolling a pipet across.
3. Lay gel top-side down and remove bubbles between gel and paper.
4. Surround the gel with Parafilm strips.
5. Soak the MagnaGraph membrane in ddH₂0 for 3 min and lay it on top of the gel.
6. Place 2 pieces of chromatography paper on top of membrane.
7. Stack at least 3 inches of cut-to-size brown paper towels on top of paper.
8. Place an oversized piece of plastic or glass on top of the towels. Weigh the whole set up down with about 400 gm (like water in tupperware).
9. Allow the RNA to transfer overnight.

1. Bake membrane in 80℃ vacuum oven for 20 min.
2. UV crosslink (on optimal setting of Fisher Crosslinker) the membrane (face side up).
3. Store between 2 pieces of chromatography paper in a plastic bag.

1. Make a 10 ng/µl dilution of the probe.
2. Add 10 µl (100 ng) of probe to a 0.5 ml screw-cap eppendorf tube.
        Adjust volume to 28 µl with water. (ie, 7 µl of probe needs 21 µl H₂ O.)
3. Boil for 5 min and then chill on ice for 5 min.
4. Briefly spin up/down in the microfuge.
5. Following the protocol from the Amersham Multiprime Kit (RPN.1601):
        
   1. Add 10 µl of Buffer (solution 1, blue cap vial)      
   2. Add 5 µl of Primer (solution 2, black cap vial)      
   3. Add 5 µl of [a-³²P]dCTP (3000 Ci/mmol)      
   4. Add 2 µl of Enzyme (solution 3, Klenow fragment)
                Final volume at this point should be 50 µl.      
   5. Incubate for 1 hr at 37℃ or overnight at room temperature.

   Prehybing the blot

   1. For hybridization in a tube, about 3-5 ml of buffer is sufficient. If a bag is used, it's
      about 0.2 ml of buffer per square cm of membrane. It is critical that the mem-
      brane be wet thoroughly.
   2. Boil 200 µg/ml of sheared salmon sperm DNA in a 50 ml conical tube for 5 min and
      chill on ice for 5 min. (If it's a total of 10 ml, use 2 mg salmon sperm.)
   3. Add the hybridization buffer (5 ml) and the formamide (5 ml) to the salmon sperm.
   4. Put the membrane in a tube and wet it with this mixture.
   5. Incubate at 42℃ for at least 30 min.

   Hybridization

   1. Check the incorporation of [32P]dCTP into the probe first.
      1. Spot 1 µl of reaction mixture onto the center of a small piece of DE81 paper
          that is placed into the bottom of a 7 ml scintillation vial. Do this twice (i.e., into two vials. One will be washed; the other will be the control.)
      2. Let the spot dry (about 2 min).
      3. Fill the wash vial with 5 ml of 0.5 M Na₂HPO₄and let it sit for 3 min. Then aspirate off the wash. Do this twice more.
      4. Add 5 ml of scintillation fluid and count.
      5. % incorporation = (cpm of washed/cpm of unwashed) x 100. Anything over 10% is usually usable. Using the kit, it's usually 40%.
   2. The specific activity (S.A.) of the probe is also important.
           S.A. = [(cpm of washed) x (volume of labelling reaction)]/µg amount of DNA
                If S.A. is over 1x10⁸, the probe is OK.
   3. Boil the probe for 5 min and chill it on ice for 5 min.
   4. Up/down spin the tube to bring down the condensation on the lid.
   5. Add it to the tube or bag.
   6. Let it hybridize overnight (at least 16 hr) at 42℃.

    

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