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ProbeCleanup
ProbeCleanup

Cleaning-up and Quantifying a Radiolabeled Probe

determine the percent incorporation of radiolabeled nucleotide:

  1. remove 1 μl of the probe solution and add to 99 μl water and mix well to make the diluted probe quantitation solution
  2. on each of two DE81 paper squares, spot 5 μl of diluted probe quantitation solution and allow to air dry completely at room temperature (about 5 or 10 minutes)
  3. place each DE81 square into a separate scintillation vial
  4. add 4 ml water to one of the vials, cap tightly and shake vigorously. this is the total radioactivity sample
  5. to the other DE81 square in a scintillation vial, add 4 ml of 200 mM Na2HPO4 solution. cap tightly and mix gently but completely. let sit with periodic gentle mixing for 5 minutes, then decant and discard the Na2HPO4 solution. do this step three times for a total of 3 Na2HPO4 rinses
    • do not go crazy with vigorous shaking -- the DE81 paper is really make out of paper, and if you shake it too hard while it's wet, it will fall apart
  6. add 4 ml water to the Na2HPO4 rinsed DE81 paper. this is the incorporated radioactivity sample
  7. count both the total radioactivity sample and the incorporated radioactivity sample in a scintillation counter on the open channel
    • using water as a fluor in this manner counts the Cerenkov radiation from the decaying 32P. it has a detection efficiency of about 60% relative to commercially available fluors. this only works on the open channel, and only with the 32P isotope. for all other isotopes, use a fluor and the appropriate channels on the scintillation counter
    • the percent incorporation is the incorporated radioactivity sample cpm divided by the total radioactivity sample cpm
      • over 50% incorporation is a good result

Calculate the specific activity of your probe:

The specific activity of the probe needs to be greater than 5 x 108 cpm / μg for efficient detection in hybridization experiments. PcrRadiolabeling can produce probes with specific activities higher than 2.0 x 109 cpm/μg

removing unincorporated nucleotides and primers: we use an Illustra ProbeQuant G-50 size exclusion column from GE Healthcare (cat #28-9034-08) for this purpose. Removing the unincorporated radionucleotides can reduce the background noise on your blot. The G-50 column removes species smaller than about 20 nt in length.

  1. resuspend the G-50 matrix by vigorous shaking
  2. open the cap of the G-50 tube 1/4 turn and snap off the bottom flange to open the column
  3. place the column in a 1.5 ml eppendorf tube and centrifuge at 735g for 1 minute
  4. discard the eluate
  5. bring the volume of the labeling reaction up to 50 μl with water if necessary, and add to the top of the column in the center of the matrix
    • be sure that the solution is not allowed to drip down the sides of the column directly without entering the matrix
  6. add the cap loosely to the column and spin the column in the eppendorf tube at 735g for 2 minutes
    • the unincorporated nucleotides and primers are trapped in the matrix of the column. the labeled probe is in the eluate
    • if you used radionucleotides with some added dye to make them easier to see and work with, the colored dye should remain completely in the column matrix
  7. discard the column. tighly cap the eppendorf tube, and put on an eppendorf lid clamp. incubate in a boiling water bath for 2 minutes
  8. place the eppendorf tube in an ice/water bath for 2 minutes. The probe can now by added directly to the hybridization solution of either one or two blots for SouthernBlotting


Comments

Isn't it wonderful to see how our kids think about these tinhgs? It feels good to see my deliberate hard work at being colorblind being the norm for my daughter.Back in the '08 election cycle, my daughter asked me what color Barack Obama was (we don't do the TV thing, hence no visual), and I panicked thinking about trying explain the racial issues involved.I started out, he's black, though it doesn't - and was sharply interrupted by her responding with , no, I mean red or blue, Mommy .
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