Precaution steps for casting a gel.

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    • #7738
      lmenwe
      Participant

      Please tell me the precaution steps for casting a gel. thanks.

    • #73297
      canalon
      Participant

      Agarose ou Acrylamide? Other?
      Ethidium bromide included or not? This is a bit vague, but to start with the most common, agarose:

      1- Melting the agarose: if in microwave be careful not to overboil (take a large flask, do not overheat, beware when handling liquid from the microwave). If you use a hot plate, the main thing to avoid is to burn the agarose.

      2- cool agarose down, nothing special, but do not cool too much (then go back to 1)

      3- If you add EtBr, it’s now time to. beware to wear gloves and goggles, work in the designated area (or in area easy to clean in case of spill). Do NOT breathe vapors. Mix well.

      4- Pour agarose into gel cast, and add comb. If EtBr is is in the gel, be carefull of spills. Before pouring make sure that your cast is leakproof (depends on the model, ask someone who has some experience with that particular model) and on a flat surface (to keep gel thickness constant. Then let cool down enough (I usually count ~10min after the gel has reached the milky color it has when it is set.

      You are ready to go.

    • #73299
      lmenwe
      Participant

      Yes it is agarose gel and Ethidium Bromide is not included. My tutor only want us to prepare gel for her and this is not the complete experiment as described in the lab manual. Thanks for your help.

    • #73321
      mith
      Participant

      Be sure to wait for the stuff to cool before adding EtBr, and also make sure to wear gloves

    • #73841
      icequeen11
      Participant

      Let the agarose cool till you can touch the flask with your bare hand, pain-free, before adding the EtBr. Swirl the flask every now and then as it’s cooling too, to avoid getting polymerizing regions against the glass.

      I was doing Southern blots; I don’t know how important it is for normal check gels and whatnot, but when casting, make sure your tray is level. Pouring agarose when it’s too hot will cause the plastic tray to warp, giving you areas of uneven thickness in the final product; this may affect migration rate and transfer.

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