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Restrictive Enzymes

  1. May 13, 2004 #1
    I'm wondering if, when put into the DNA together, two different restriction enzymes will interfere with each other, and how. Will two different restriction enzymes cut the DNA as they each would separately, since they are specifically formed to react with it and not each other, or would they disturb each other? Or, is this a case-by-case issue?
     
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  3. May 13, 2004 #2

    iansmith

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    There is usually no problem when using two restriction enzyme in the same reaction. It will only be a problem if you restriction site of you two enzymes overlap. The major problem is usually the buffer used. The condition of the reaction will affect the effcienty of the enzyme.
     
  4. May 14, 2004 #3

    Monique

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    You need to be careful that you don't add more enzyme than 10% of the reaction, the glycerol will inhibit the cutting. There shouldn't be interference, though Ian makes a good point that there might be a problem if the two restriction sites overlap, it wouldn't be smart wanting to cut DNA with such like enzymes anyway, so I don't think that will ever be the case :)

    Be carefull that the buffer conditions are the same, adjust the amount of enzyme if one enzyme cuts less efficiently in a certain buffer.
     
  5. May 14, 2004 #4
    I agree with Ian, as two restriction enzymes could cancel each other out, or, worse, cut each other up instead, and then you'd have a whole big (relatively :wink: ) mess to clean up. As long as the restriction sites don't overlap or are too near each other, then using two or more different enzymes shouldn't present too much of a problem.









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    Last edited: May 14, 2004
  6. May 14, 2004 #5

    Monique

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    Restriction enzymes don't cut eachother. Double digests are perfectly safe if both enzymes work in the supplied buffer. For some reason there are exceptions which can be found on the following site http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp I think that is because of buffering conditions and not recognition sequence competition.

    ps. glycerol should be less than 5% to prevent star activity.
     
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