lab-protocols

SPRI Bead DNA Extraction Protocol

Reagents

Equipment

Procedure

1) Lysis

  1. Prepare lysis master mix containing 5 µL of 20 mg/mL proteinase K and 295 µL proteinase K buffer solution per sample plus a little extra to account for pipetting error.
  2. Add 300 µL of lysis master mix to a 1.5 mL microcentrifuge tube containing up to 20 mg of sample tissue (approximately the size of a grain of rice).
  3. Incubate samples at 55 °C in shaker incubator set to 400 for at least 4 hours or overnight.

2) Isolation

  1. Allow Speedbead solution to warm to room temperature and mix by gently inverting the solution. Mix until beads are no longer sticking to the bottom of the tube. Before using beads, allow any bubbles that have been introduced by mixing to escape from the solution.
  2. Add 300 uL of bead solution to tube containing lysis product.
  3. Mix sample by flicking tube.
  4. Briefly centrifuge to collect sample in bottom of tube.
  5. Let sample sit for 5 minutes.
  6. Place sample on magnet for 5 minutes or until all beads have collected on magnet and solution is clear.
  7. Remove and dispose of solution without disturbing the beads on the magnet.
  8. Add 1 mL of 70% ETOH.
  9. Let sit for 1 minute.
  10. Remove and dispose of ethanol without disturbing the beads on the magnet.
  11. Add 1 mL of 70% ETOH.
  12. Let sit for 1 minute.
  13. Remove and dispose of ethanol without disturbing the beads on the magnet.
  14. Briefly centrifuge sample at low speed to collect residual ethanol and return to magnet. Beads should remain in place on side of tube.
  15. Remove and dispose of remaining ethanol without disturbing the beads on the magnet.
  16. Add 100 µL of TLE buffer and flick tube to mix beads into solution until they are no longer sticking to side of tube.
  17. Briefly centrifuge to collect solution in bottom of tube.
  18. Let stand for 1 minute.
  19. Place on magnet stand for 5 minutes.
  20. Remove as much of solution as possible without disturbing beads and put into new labelled tube.

Comments

To increase yield it may be beneficial to increase the time sample is allowed to sit at step 18. Warming the sample during this step may also help.