Arichard:Protocols/size selection 150402

From ZhangLabWiki
Jump to navigation Jump to search

MDA amplicon size selection[edit]

Prep[edit]

  • For each sample pool:
    • Place a 0.5 ml tube inside a low binding 1.5 ml tube. Use a 20 G needle to punch a hole in the bottom of the low binding 0.5 ml tube, then UV treat.
    • UV 400 ul TE buffer.
    • UV a nanosep tube.
    • UV 1 ml 100% EtOH, 40 ul 3 M NaAc, and 1 ul GlycoBlue in a 1.5 ml tube.
    • Chill 75% EtOH wash at -80 C before using.

Protocol[edit]

Size selection[edit]

  1. Run pools up to 40 ul on 5 lane PAGE gels, 250 V for 20 minutes. Use 20 ul 6X Loading Dye (NEB), 2 ul Low Mass Ladder (Invitrogen). Use additional 0.5X TBE to equalize volumes between all pools.
  2. Add 3 ul SYBR Gold to tray with 100 mL 0.5X TBE. Mix well before adding gel to avoid bright spots.
  3. Incubate gel 2 minutes in tray on shaker.
  4. Size select between 200 and 800 bp with scalpel. Load gel fragment into 0.5 ml tube (inside 1.5 ml tube).
  5. Spin gel in tube-in-tube setup 2 minutes at 12,000 RPM. Remove and discard 0.5 ml tube.
  6. Add 400 ul TE buffer and incubate shaking for 1 hour at 37 C.
  7. Load into NanoSep tube and spin 3 minutes at 15,000 RPM.

Ethanol precipitation[edit]

  1. Load Nanosep flowthrough into EtOH precipitation mix and incubate at -80 C for 30 minutes.
  2. Spin for 30 minutes at 10,000 RPM at 4 C. Remove and discard supernatant.
  3. Wash with 750 ul cold 75% EtOH and spin 5 minutes at 10,000 RPM at 4 C.
  4. Remove wash and let dry 5-10 minutes. Resuspend in 20 ul H2O. Store at -20 C until sequencing.