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Trouble-shooting: Low yield

  • 72C for 20 seconds for fragments < 500 bp
  • 72C for 40 seconds for fragments 1.2 bp.
  • Suggestion:

    Longer times, not higher temperatures should be used when longer templates or suspected secondary structure is present.

    Back to Top

    Enzyme activity is low

    For Roche Applied Science polymerases, 100% activity is guaranteed through the control date.

    Suggestions:

    1. Check the control date. If necessary, obtain fresh enzyme
    2. Try "Hot start" technique to help retain activity through thermal cycling
    Back to Top Cycle number is too high

    Most templates require 25-30 cycles.

    Suggestion:

    Cycle number should be based on starting concentration of template DNA.

    If the number of target molecules in your sample is...

    Then we recommend the following number of Cycles... 3 x 105 25-30 1.5 x 104 30-35 1.0 x 103 35-40
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    TAG: Trouble shooting Low yield

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