Matt:LabNotes/2014-8-7

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Revision as of 02:32, 8 August 2014 by >Mzcai (→‎EtOH Precipitation)
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Agi26k0gap Probe Production Continued

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 12 tubes at -80C for 3 hrs
  • Spun 12 tubes at 10,000rpm at 4C for 30min
  • Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min
  • Discard supernatant and let dry in hood for 10min
  • Resuspend each tube with 10ul (120ul total)