Matt:LabNotes/2013-5-17

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Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-16

PAGE-Size Selection of 20 gap probes[edit]

  • Noi said that the band on the previous TBU gel looked relatively faint and recommended putting 3ug of DNA per well instead of the usual 2ug to increase yield
  • I will run 3 gels each with 60ul (~3.4 ug of DNA each)

Sample

Components 1X Volume 3X Volume
Enzyme digested probes (~10ug/180ul) 55 165
TBE-Urea Buffer 2X 55 165
Water 0 0
Total 110 330

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
  • Cut out band below 200bp

Gel 1:
File:2013-05-17 Gel1SizeSelection 20gap.jpg

Gel 2:
File:2013-05-17 Gel2SizeSelection 20gap.jpg

Gel 3:
File:2013-05-17 Gel3SizeSelection 20gap.jpg

  • 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


Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-21