Daniel:Notebook/ComboLock/2017-5-2
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Production Run (Started Friday 4/28)[edit]
Library Prep[edit]
- Qiaquick Purification
- Combine 20 uL each sample into a 1.5 mL tube following matrix
- Add 200 uL (5X) PB (binding buffer) to sample
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
- Repeat above step with 400 uL PE buffer
- Dry spin column for 2 minutes at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Add 30 uL nfH2O to column
- Spin for 1 minute at 14000 rpm
- Size Select TBE Gel
- Mix 30 uL 10X TBE and 24 uL 6X dye
- Aliquot 9 uL for each sample/ladder into a separate tube
- Add 15 uL sample or 2.5/12.5 uL ladder/10X TBE to aliquot tubes
- Mix and add 23 uL per lane to gel
- Run gel at 230V for 24 minutes
- Add 2 uL SYBR gold and incubate on shaker for 3 minutes
- Rinse and image in gel doc
- Cut out bands at approximately 221bp
- 2017-05-02-ProductionRun-SizeSelection.png
Pre Selection
- 2017-05-02-ProductionRun-SizeSelection-After.png
Post Selection
- Add extracted gel bands to 0.5mL tube with 0.2 gauge needle hole inside a 1.5 mL tube
- Centrifuge for 1:30 at 13000rpm
- Throw away 0.5mL tube and add 500 uL TE buffer to each tube
- Incubate at 37C for 1 hour
- Centrifuge for 1:30 at 13000rpm
- Withdraw supernatant and add it to a nanosep column
- Centrifuge for 1:30 at 13000 rpm
- Remove flow through and add it to new 2 mL tube (~500 uL)
- Ethanol Precipitation
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1 uL glycoblue to each tube
- Incubate overnight at -80C
Tube | Sample 1 | Sample 2 | Indexes |
1X | 1AX | 1BX | 20 |
5AX | 5AX | nfH2O | 24 |
1Y | 1AY | 1BY | 25 |
5AY,4AY | 5AY | 4AY | 26,27 |
Continued tomorrow