Lab Work

Table of Contents

Each Morning After Trapping

Remember the lab grab bag, goggles, and keys! For all vials cover the written label with tape to prevent the label from being rubbed off. Hair samples and FTA cards can be stored at room temperature. Serum separator tubes must be centrifuged for 10 minutes, then they and anticoagulant tubes will be divided and placed in the -80 °C Freezer. Fecal, parasite, and buccal samples can be stored in the -20 °C freezer.

Running Samples Later - DNA Extraction

  • FTA cards can be stored for months or years at room temperature. It is better to store at room temperature than freeze.

Need:

  • Qiagen DNeasy Blood and Tissue Kit
  • PBS Buffer

Directions

DNA Extraction

  • Cut a sample from the FTA card with contaminant free razor blades, small enough to fit in the bottom of a microcentrifuge tube
  • Use 20ul protenase K and 200ul PBS buffer, vortex and lyse overnight at 56C. If using liquid blood only add enough PBS buffer to make a total 220ul.
  • The next day vortex again.
  • Add 200ul Buffer AL. Lysis buffer detergent-this dissolves lipids.
  • Incubate at 56C for 10 minutes
  • Makes a film, vortex again
  • Add 200ul 96-100% ethanol and vortex
  • Pipette to spin columns (should be 620ul solution), centrifuge at 8,000 rpm(6,000g) for 1min. DNA will stick to charged membranes.
  • Discard flow through and transfer to a new collection tube.
  • Add 500ul AW1 wash buffer.
  • Centrifuge again, 8,000 rpm for 1 minute
  • Dispose of liquid and collection tube
  • Wash with 500ul buffer AW2, centrifuge 14,000 rpm 3 min
  • Dispose of liquid and transfer to new tube
  • Add 200ul buffer AE- elution buffer, neutralizes the membrane. Sit at room temp for 5 minutes.
  • Centrifuge again 8,000 rpm for 1 minute.
  • DNA is in the solution. Repeat previous step for better yield.
  • Discard membrane.
  • DNA extracted, can store for years

Spectrophotometer

  • Run two blanks of just the buffer solution
  • Then run samples 8 1.80 is perfect DNA purity. 1.7-2.0 good, lower may still be OK, depends on the type of PCR. Negative number- concentration may be too low. Evaporate.
  • Can dilute to remove contaminants. Need only 1 microliter of DNA, so it can also be diluted

Now ready for PCR!

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