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    • #7823
      icequeen11
      Participant

      Hey all,

      I just performed a primary screen of a genomic library. My films look spotty, but not in the same mottled way as the plated phage plaques, more random. I did duplicate lifts, and while both films are spotty to the same degree, few (if any) of the spots overlap.

      Going back to the procedure, I boiled the labeled probe (32P dCTP-labelled cDNA) for 5 minutes, then put it on ice for a few minutes (to minimize renaturation) and added 1 mL hybridization buffer (6x SSC, 10x Denhardt’s, 2% SDS, 0.2 mg/mL sheared calf thymus DNA as a blocker). However, being on ice, the SDS and SSC precipitated out.

      I added this precipitated solution to my hybridization, on the assumption that upon incubating at 60 degC overnight, it would go into solution and every would go merrily along.

      Upon seeing my films, is it possible that the probe precipitated out with the other stuff and stuck to my membranes in chunks, never to come off again? I washed down to 0.5x SSC + 1% SDS, and there’s minimal background behind the spots after an 18h exposure. Therewas no signal after 2h exp. Has anyone else had this issue? Comments?

      Cheers!

    • #73910
      blcr11
      Participant

      It sounds to me like you’ve got background hybridization only–nothing bound except a small random amount of probe. Now, whether or not that happened because the SDS and salts precipitated, I don’t really know. Did everything go back into solution during the hybridization? Perhaps you could have re-heated the probe mix while it was on the membrane? Better not to precipitate anything, I suppose.

      If you take and dump your denatured carrier/probe mix directly into the hybridization mix, seal everything, and then proceed to the hybridization temp (usually around 60-65C) I wouldn’t think you’d have that much trouble with snap-back hybridization. I don’t think you need to put the probe mix on ice.

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