Matt:LabNotes/2014-6-23

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Revision as of 23:08, 24 June 2014 by >Mzcai (Created page with "===Size Selection=== *Ladder: 0.5ul Low Mass Ladder + 2.5ul H2O + 2ul Loading Dye *Samples: 5ul Sample + 2ul Loading Dye *Gel 1: Ladder, Ran Hex, Poly(dT), FISSEQ_RT *Gel 2: ...")
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Size Selection

  • Ladder: 0.5ul Low Mass Ladder + 2.5ul H2O + 2ul Loading Dye
  • Samples: 5ul Sample + 2ul Loading Dye
  • Gel 1: Ladder, Ran Hex, Poly(dT), FISSEQ_RT
  • Gel 2: Top 48, No Enzyme, Nextera +, Ladder
  • 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 1.5 mL tube below with pipette tip.
  • Added 450 ul of 1X TE buffer
  • Vortexed for 45-60 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 (volume ~ 400ul/ tube)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 6 tubes at -80C overnight