Matt:LabNotes/2014-3-5

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Agi26k0gap Padlock Probe Prep[edit]

Procedure[edit]

Sample

Components 1X Volume 3X Volume
Enzyme digested probes (~10ug/180ul) 58 174
TBE-Urea Buffer 2X 58 174
Water 0 0
Total 116 348

3 Low Mass DNA Ladders

Components 3X Volume
Low Mass DNA ladder (1 ug/ul) 3
TBE-Urea Buffer 2X 15
H2O 12
Total 30
  • Ran the 3 gels at 220V for 30min
  • Shake in SYBR Gold for 6+ minutes
  • Cut out band below 200bp

Gel 1:
File:2014-03-05 Agi26k0gapPrep Gel1.jpg

Gel 2:
File:2014-03-05 Agi26k0gapPrep Gel2.jpg

Gel 3:
File:2014-03-05 Agi26k0gapPrep Gel3.jpg

  • Put cut out gel in 0.5mL tube (with hole 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 3.5hr 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

Day 2[edit]

Ethanol Precipitation after PAGE size selection

  • Spun 6 tubes in -80C at 10,000rpm at 4C for 30min
  • Discard supernatant and add 650ul 75% EtOH to wash and spin at 14,000rpm at 4C for 5 min
  • Discard supernatant and let dry in hood 10 min
  • Resuspended each of the 6 tubes with 10ul H2O (60ul total)

TBU Gel Quantification Plan

  • Dilute 2ul into 20ul
  • Mix 1:1 with 2X buffer (20X dilution)
  • Load sample wells with 2, 4, 6, 8, and 10 ul
  • Load Low Mass DNA ladders in 0.5, 1, and 2 ul
' 0.5 ladder 1 ladder 2 ul 4 ul 6 ul 8 ul 10 ul 2 ladder
DNA volume 0.5 1 0.1 0.2 0.3 0.4 0.5 2
H2O 1.5 1 0.9 1.8 2.7 3.6 4.5 0
2X Buffer 2 2 1 2 3 4 5 2
Total 4 4 2 4 6 8 10 4

File:2014-03-07 Quantify Agi26k0gap.jpg

  • Intensity to [DNA] calculations
  • 20X diluated sample concentration is 1.64ng/ul
  • Starting sample concentration: 32.8ng/ul -> total yield about 1.9ug/(193nt*325 Da/nt) = 30.3 pmole