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30 Preparation

 

Review the steps below to learn about the DNA extraction kit.

 

Procedure

Use the Protocols.io copy of the procedure accessed here to run and check off each step of the procedure.

 

Note Before You Begin: 

Review the complete protocol before beginning. Several of the steps in this procedure are time and/or temperature-sensitive, so it’s important that you know what to expect and how to manage your time. There are also several types of tubes with specific names that need to be used for specific steps of the protocol. They are shown in the illustration below to help you keep track of these materials.

 

Safety

All plastics and cultures will be treated as biohazards. Cultures will be treated with 10% (final) bleach overnight. Plastic tubes, pipettes, and spin columns used for the extraction process will be disposed of in the biohazard containers.

 

Supplies

We will need several supplies for this lab, including enzymes.

 

Materials

200 µl liquid overnight culture (~1 x 108 – 1x 109 cfu/ml) or 1/8 of a 10 µl loop of colonies from a plate

 

Consumables

  • Lysozyme human (Sigma, L1667)
  • Trizma® hydrochloride solution (Sigma, T2819)
  • 5 M sodium chloride solution (Sigma, S6546)
  • SDS at 10% v/v (Sigma, 71736)
  • Phosphate Buffered Saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
  • TE buffer (Sigma, 8890-100ML)
  • Proteinase K (NEB, P8200AAVIAL) from the Monarch Spin gDNA Extraction Kit
  • Monarch® RNase A (NEB, T3018-1) from the Monarch Spin gDNA Extraction Kit
  • CTAB buffer (Promega, MC1411)
  • Monarch® gDNA Elution Buffer (NEB, T3016-1) from the Monarch Spin gDNA Extraction Kit
  • Monarch® gDNA Binding Buffer (NEB, T3014-1) from the Monarch Spin gDNA Extraction Kit
  • Monarch® Spin Collection Tubes (NEB, T2118-1) from the Monarch Spin gDNA Extraction Kit
  • Monarch® Spin Columns S2C and Tubes (NEB, T3017-1) from the Monarch Spin gDNA Extraction Kit
  • Monarch® gDNA Wash Buffer (NEB, T3015-1) from the Monarch Spin gDNA Extraction Kit
  • Qubit 1x dsDNA HS Assay Kit (ThermoFisher, Q33230)
  • Nuclease-free water (e.g., ThermoFisher, AM9937)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 5 ml Eppendorf DNA LoBind tubes

 

Equipment

  • Eppendorf 5424 centrifuge (or equivalent)
  • Eppendorf Thermomixer
  • Vortex mixer
  • P1000 pipette and filter tips
  • P200 pipette and filter tips
  • P100 pipette and filter tips
  • P20 pipette and filter tips
  • P10 and P2.5 pipette and filter tips

 

License

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Biotechnology and Sustainability Copyright © by Carlos Goller is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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