Arichard:Protocols/MDA amplicon size selection: Difference between revisions
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>Andrew (Created page with "==MDA amplicon size selection== ===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. U...") |
>Andrew |
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===Prep=== | ===Prep=== | ||
* For each sample | * For each sample pool: | ||
* Set up a nanosep tube | ** 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 | ** 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]
- 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[edit]
- 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.