This experiment is part of the iGEM 2019 Challenge Challenge Grant. Browse more projects

Transforming Styrofoam waste into biodegradable plastic

By Alec Brewer, Jermaine Elijah Austin, Katie Zhang, Simonne Guenette, Shaalini Desai, Benjamin Ascoli, Kobe Rogers, Jainam Modh, Hannah Towler, Evan Biedermann, and Aarati Pokharel
Backed by Richard Davies, Melinda Rogers, Karen Bruce, James Cooper, Janice Hawn, Morgan Hough, Elizabeth Nestor, Jennifer Kreinheder, Mary Hao, Shaun Masavage, and 28 other backers
University of Virginia
Charlottesville, Virginia
BiologyEngineering
DOI: 10.18258/13643
Grant: iGEM 2019 ChallengeGrant: iGEM 2019 Challenge
$1,747
Raised of $1,550 Goal
112%
Funded on 7/10/19
Successfully Funded
  • $1,747
    pledged
  • 112%
    funded
  • Funded
    on 7/10/19

Restriction Enzyme Digest

Procedure:

  1. For 50 uL Reaction

    1. 1 ug of DNA 

    2. 5uL of respective NEB Buffer (10X)

    3. 1uL of enzyme 1

    4. 1uL of enzyme 2

    5. Remaining should be MilliQ Water

  2. In rxn tube, mix the following order: 1. Water; 2. Buffer; 3. DNA; 4. Enzymes

  3. Incubate at 37C for 30 minutes (If you have time save enzymes otherwise incubate for 1hr) then heat kill at 80C for 20 mins

    1. Note: heat kill temperature will depend on restriction enzymes, check the enzyme heat kill temperatures

  4. If you are worried about the backbone reannealing, treat the backbone with Shrimp Alkaline Phosphatase (rSAP):

    1. rSAP can be added during the digest (0.5ul)

    2. rSAP can also be added after the digest:

      1. 1 pmol of DNA ends; 1pmol of DNA ends is about 1ug of 3kb ladder

      2. 2ul of 10X Cutsmart Buffer

      3. 1 unit of rSAP (if you have the 500 unit tube, then add 0.5ul)

      4. MilliQ Water up to 20ul

      5. Incubate at 37C for 30 mins

      6. Heat Inactivate at 65C for 5 mins

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