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    • #12151
      cmgross
      Participant

      I need to learn at least 2 different ways to determine if a 100 kb ds DNA viral genome is circular or linear. We are starting with highly purified viral DNA in a tube. I know that circular and linear DNA run differently on a gel, but I don’t think you can run fragments that big on an agarose gel. Thanks

    • #94288
      canalon
      Participant

      Pulse field gel electrophoresis (PFGE) is your friend

    • #94290
      JackBean
      Participant

      By restriction enzymes you could recognise it (circular = you will get as many fragments, as many times you cut; linear = you get +1 fragment), but I guess ou don’t have restriction map, do you? 🙂

    • #94298
      qqsvery
      Participant

      U can try density gradient centrifugation method!good luck!

    • #95894
      michellethemit
      Participant

      Disregard this, the method I was going to suggest required running on an agarose gel, and the OP mentioned that he was unsure whether a 100kb size genome would run properly.

    • #95974
      Darwin420
      Participant

      Yea, pulse field is indeed your friend of big samples, if you do this you should be able to differentiate a circular from a linear DNA sample.

      Or set up a density gradient centrifugation.

      I can’t think of any more really.

    • #106045
      gradkid
      Participant

      Run 2 restriction digests with 2 different restriction enzymes, followed by a combination digest. Let’s say enzyme A cuts your DNA into 3 fragments and enzyme B cuts it into 4. You don’t know if your DNA is linear with 2 restriction sites for A and 3 sites for B or circular with 4 sites for A and 5 sites for B. However, if you run a combination digest with enzyme A+B, you can determine if it’s linear or circular. If the combination digest has 7 fragments, it’s circular; if it has 6 fragments, it’s linear.

      I think.

    • #106295
      aptitude
      Participant

      Pulse-field gel electrophoresis is good for large DNA fragments.

    • #106307
      greatmicrobiologist
      Participant
      quote michellethemit:

      Disregard this, the method I was going to suggest required running on an agarose gel, and the OP mentioned that he was unsure whether a 100kb size genome would run properly.

      Support you.


      @Cmgross
      :

      before going for agarose gel elctrophoresis or polyacrylamide gel electrophoresis, go for PCR amlification for your desired quantity.

    • #107051
      JackBean
      Participant
      quote aptitude:

      Pulse-field gel electrophoresis is good for large DNA fragments.

      yeah, that’s what the first reply suggested

      quote greatmicrobiologist:

      quote michellethemit:

      Disregard this, the method I was going to suggest required running on an agarose gel, and the OP mentioned that he was unsure whether a 100kb size genome would run properly.

      Support you.


      @Cmgross
      :

      before going for agarose gel elctrophoresis or polyacrylamide gel electrophoresis, go for PCR amlification for your desired quantity.

      Why should he run PCR? How will that help?

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