Arichard:Protocols/MDA amplicon size selection: Difference between revisions

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>Andrew
 
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===Prep===
===Prep===


* For each sample, place a 0.5 ml tube inside a 1.5 ml tube. Use a 20 G needle to punch a hole in the bottom of the 0.5 ml tube. UV treat.
* For each sample pool:
* Set up a nanosep tube for each sample.
** 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 treat 1 ml 100% EtOH, 40 ul 3 M NaAc, and 1 ul GlycoBlue in each 1.5 ml tube, 1 tube per sample.
** UV treat 400 ul TE buffer for each pool.
** Set up and UV treat a nanosep tube.
** Mix and UV treat 1 ml 100% EtOH, 40 ul 3 M NaAc, and 1 ul GlycoBlue in a 1.5 ml tube.


===Protocol===
===Protocol===


====Size selection====
====Size selection====
# Pool similar samples (keep bacteria separate from mammalian samples), up to 2 samples per pool. Match libraries with similar intensities on the analysis gel to avoid excessive imbalance between libraries.
# Run whole pools on 5 lane PAGE gels, 250 V for 22 minutes. Use 10 ul dye, 2 ul low mass ladder. Use additional 0.5x TBE to equalize volumes between all pools.
# Add 6 ul SYBR Gold to tray with ~1 cm 0.5x TBE, mix well.
# Incubate gel 2 minutes in tray with SYBR Gold on shaker.
# Size select between 300 and 600 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 degC.
# Load into nanosep tube and spin 3 minutes at 15,000 RPM.


====Ethanol precipitation====
====Ethanol precipitation====
# Load nanosep flowthrough into EtOH precipitation mix and incubate at -80 degC for 20 minutes.
# Spin for 25 minutes at 10,000 RPM at 4 degC. Remove and discard supernatant.
# Wash with 750 ul 75% EtOH and spin 5 minutes at 10,000 RPM at 4 degC.
# Remove wash and let dry 5-10 minutes. Resuspend in 20 ul H2O. Store at 4 degC until sequencing.

Latest revision as of 00:04, 7 September 2013

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 treat 400 ul TE buffer for each pool.
    • Set up and UV treat a nanosep tube.
    • Mix and UV treat 1 ml 100% EtOH, 40 ul 3 M NaAc, and 1 ul GlycoBlue in a 1.5 ml tube.

Protocol[edit]

Size selection[edit]

  1. Pool similar samples (keep bacteria separate from mammalian samples), up to 2 samples per pool. Match libraries with similar intensities on the analysis gel to avoid excessive imbalance between libraries.
  2. Run whole pools on 5 lane PAGE gels, 250 V for 22 minutes. Use 10 ul dye, 2 ul low mass ladder. Use additional 0.5x TBE to equalize volumes between all pools.
  3. Add 6 ul SYBR Gold to tray with ~1 cm 0.5x TBE, mix well.
  4. Incubate gel 2 minutes in tray with SYBR Gold on shaker.
  5. Size select between 300 and 600 bp with scalpel. Load gel fragment into 0.5 ml tube (inside 1.5 ml tube).
  6. Spin gel in tube-in-tube setup 2 minutes at 12,000 RPM. Remove and discard 0.5 ml tube.
  7. Add 400 ul TE buffer and incubate shaking for 1 hour at 37 degC.
  8. 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 degC for 20 minutes.
  2. Spin for 25 minutes at 10,000 RPM at 4 degC. Remove and discard supernatant.
  3. Wash with 750 ul 75% EtOH and spin 5 minutes at 10,000 RPM at 4 degC.
  4. Remove wash and let dry 5-10 minutes. Resuspend in 20 ul H2O. Store at 4 degC until sequencing.