Matt:LabNotes/2014-6-27: Difference between revisions
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>Mzcai (Created page with "==Agi26k0gap Probe Production Continued== *Continued from: Matt:LabNotes/2014-6-24 ===PAGE Size Selection=== *Run 4 gels of ~3.2ug ssDNA in each **Corresponds to 22.5ul ...") |
>Mzcai |
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*Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT. | *Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT. | ||
*Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip. | *Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip. | ||
*Added | *Added 900 ul of 1X TE buffer | ||
*Vortexed for | *Vortexed for 60 min at 37 C in incubator | ||
*Centrifuged at 15,000 rpm for 3 min at RT | *Centrifuged at 15,000 rpm for 3 min at RT | ||
*Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min | *Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min | ||
*Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min | *Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min | ||
*Transfered spnt to fresh 1.5 mL tube | *Transfered spnt to fresh 1.5 mL tube | ||
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2 | *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2 | ||
*Vortexed and placed the 4 tubes at -80C | *Vortexed and placed the 4 tubes at -80C |
Revision as of 21:48, 27 June 2014
Agi26k0gap Probe Production Continued
- Continued from: Matt:LabNotes/2014-6-24
PAGE Size Selection
- Run 4 gels of ~3.2ug ssDNA in each
- Corresponds to 22.5ul
Components | 1X Volume | 4X Volume |
Enzyme digested probes | 22.5 | 90 |
TBE-Urea Buffer 2X | 50 | 200 |
Water | 27.5 | 110 |
Total | 100 | 400 |
Components | 4X Volume |
Low Mass Ladder | 4 |
TBE-Urea Buffer 2X | 20 |
H2O | 16 |
Total | 40 |
- Ran at 220V for 30min
- Cut out brightest band below 200bp
EtOH Precipitation
- Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
- Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip.
- Added 900 ul of 1X TE buffer
- Vortexed for 60 min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min
- Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transfered spnt to fresh 1.5 mL tube
- Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
- Vortexed and placed the 4 tubes at -80C