Daniel:Notebook/ComboLock/2017-6-19: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 31: | Line 31: | ||
<li>Cut out bands at approximately 221bp</li> | <li>Cut out bands at approximately 221bp</li> | ||
<gallery perrow=2 heights=300px widths=300px> | <gallery perrow=2 heights=300px widths=300px mode=packed-hover> | ||
File:2017-06-19-AntibodyTest-CS-SizeSelect.png|Pre Selection | File:2017-06-19-AntibodyTest-CS-SizeSelect.png|Pre Selection | ||
File:2017-06-19-AntibodyTest-CS-SizeSelect-After.png|Post Selection | File:2017-06-19-AntibodyTest-CS-SizeSelect-After.png|Post Selection |
Revision as of 19:25, 19 June 2017
Production Run (Started Wednesday 5/8)
Library Prep
- 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
- 2017-06-19-AntibodyTest-CS-SizeSelect.png
Pre Selection
- 2017-06-19-AntibodyTest-CS-SizeSelect-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
Continued tomorrow