Matt:LabNotes/2014-8-7

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Agi26k0gap Probe Production Continued[edit]

PAGE Size Selection[edit]

  • 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[edit]

  • 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)

TBU Gel Quantification[edit]

  • Diluted Agi26k0gap 10X
    • 2ul Agi26k0gap + 8ul H2O + 10ul 2X TBE-Urea buffer
  • Made DNA Low Mass Ladder with 0.5, 1, and 2 ul
Lane 1 2 3 4 5 6 7
Sample 0.5ul DNA Ladder 1ul DNA Ladder 2ul Sample 4ul Sample 6ul Sample 8ul Sample 2ul DNA Ladder

File:2014-08-08 Agi26k0gapGelQuant.jpg

  • Agi26k0gap Concentration: 38.32 ng/ul
  • Total Mass: 38.32 ng/ul * 120ul = 4.60 ug
  • Total Amount: 4.60ug /(193nt*325 Da/nt) = 73.3 pmole (611nM)