QIAGEN Purification of PCR Products

QIAGEN Purification of PCR Products

  1. Add 500 µl QP buffer to the 500 µl DNA sample (v/v;1/1).
  2. Apply 0.8 ml QP buffer onto a Quigen-Spin20 column (to equilibriate)
  3. Spin (10K, 1min) the column with 2.0 ml MFT, and discard the filtrate.
  4. Add the mixed DNA sample into spin column.
  5. Spin (10K, 1min) and discard the filtrate.
  6. Wash with 0.8 ml QB buffer three time.
    If DNA is >1µg and 10% loss of DNA is not essential.
  7. Add 0.8 ml QF buffer into the column to elute the DNA.
  8. Spin (10K, 1min) into a clean 2 ml MFT and discard the column.
  9. Add 0.8 volumn (Appr. 0.6 ml) of icy isopropanol., mix and spin (30 min, 1.4K).
  10. Wash the DNA pellet with 0.8 ml 70 % EtOH.
  11. Dry the DNA pellet (2 min) under vacuum and redissolve in TE.
    If DNA is <1 µg and high recovery is essential,
  12. Add 0.8 ml QXE buffer to elute the DNA.
  13. Spin (10K, 1min) into a fresh 2 ml MFT.
  14. Voltex QIAEX (15 sec) until a homogeneous suspension is obtained.
  15. Immediately, add 10 µl QIAEX into the eluted DNA, mix and incubate (10 min, room temp). Voltex the sample every 2 min to keep QIAEX in suspension in order to obtain complete adsorption.
  16. Spin (30 sec, 8K) and remove the supernatent with a pipet.
  17. Wash the pellet with 0.5 ml QX3 twice.
  18. Spin (30 sec) again and remove any trace of EtOH with a pipet.
  19. Dry the pellet (2 min) under vacuum.
  20. Resuspend the pellet by voltexing in at least 20 µl TE.
  21. Incubate the suspension (5 min, room temp).