Matt:LabNotes/2014-6-27
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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