Matt:LabNotes/2014-8-7
Jump to navigation
Jump to search
Agi26k0gap Probe Production Continued
- Continued from: Matt:LabNotes/2014-8-4
PAGE Size Selection
- Run 6 gels of ~2.8ug ssDNA in each
- Corresponds to 20.7ul in each gel
Components | 1X Volume | 6X Volume |
Enzyme digested probes | 20.7 | 124 |
TBE-Urea Buffer 2X | 50 | 300 |
Water | 29.3 | 176 |
Total | 100 | 600 |
Components | 6X Volume |
Low Mass Ladder | 6 |
TBE-Urea Buffer 2X | 30 |
H2O | 24 |
Total | 60 |
- 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 12 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 (~450ul per tube)
- Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
- Vortexed and placed the 8 tubes at -80C