restriction reaction on agarose gel slices

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    • #16532
      dustman
      Participant

      I’m thinking of reducing steps for insert production.

      ATM I have following steps:
      1. PCR
      2. agarose gel with PCR products
      3. gel extraction of insert of interest
      4. restriction reaction
      5. enzyme removal <- I often use KpnI/BamHI and can’t go with heat inactivation
      6. ligation with backbone and transformation

      I know that restriction can be performed on agarose slices but never done it myself. This would save a bit of time and reagents, plus avoid DNA losses in step 5.

      Anyone got first-hand experience with this? If ‘yes’, link to a protocol would be greatly appreciated.

    • #111471
      canalon
      Participant

      Look any DGGE protocol. In my experience it does not save time at all. And neither does it saves n reagents, but buffers are cheap anyway.

    • #111567
      citroenboom
      Participant

      Why use gel extraction? Often EtBr seems to inhibit ligation. You could use PCR purification kits. Much faster!
      Or otherwise go for the good old phenol extraction (Sambrook). Quick, but dirty 🙂

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