Daniel:Notebook/ComboLock/2017-7-10
Jump to navigation
Jump to search
Oligo+Protein Test (Started Thursday July 6)
Library Prep
- Qiaquick Purification
- Combine samples A+B for each condition into a 1.5 mL tube
- Add 600 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
- Transfer column to a new 1.5 mL eppendorf tube
- Add 50 uL TE to column
- Spin for 1 minute at 14000 rpm
- Size Select TBE Gel
- Mix 30 uL sample and 6 uL 6X dye per sample
- Mix 26 uL TE, 4 uL ladder, and 6 uL 6X dye per sample
- Aliquot 36 uL for each into a separate lane
- 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
- 2017-07-10-Oligo-and-Ab-SizeSelect.png
Before
- 2017-07-10-Oligo-and-Ab-SizeSelect-After.png
After
- Note that this is a pretty bad gel, so I had to take wider fractions than I would have liked. We'll see how the sequencing results look
- 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
Continued tomorrow