Arichard:Protocols/size selection 150402
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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]
- 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.
- Add 3 ul SYBR Gold to tray with 100 mL 0.5X TBE. Mix well before adding gel to avoid bright spots.
- Incubate gel 2 minutes in tray on shaker.
- Size select between 200 and 800 bp with scalpel. Load gel fragment into 0.5 ml tube (inside 1.5 ml tube).
- Spin gel in tube-in-tube setup 2 minutes at 12,000 RPM. Remove and discard 0.5 ml tube.
- Add 400 ul TE buffer and incubate shaking for 1 hour at 37 C.
- Load into NanoSep tube and spin 3 minutes at 15,000 RPM.
Ethanol precipitation[edit]
- Load Nanosep flowthrough into EtOH precipitation mix and incubate at -80 C for 30 minutes.
- Spin for 30 minutes at 10,000 RPM at 4 C. Remove and discard supernatant.
- Wash with 750 ul cold 75% EtOH and spin 5 minutes at 10,000 RPM at 4 C.
- Remove wash and let dry 5-10 minutes. Resuspend in 20 ul H2O. Store at -20 C until sequencing.