Matt:LabNotes/2013-5-29

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Page Size Selection of 20 gap probes[edit]

  • Noi prepared some 0 gap and 20 gap probes
 *20 gap: 12.6ug (31.7ng/ul) ~410ul
 *0 gap: 11.36ug (28.4ng/ul) ~410ul
  • Ran TBU gel at 220V for 30 min with samples and ladder prepared below
    • Heated TBE buffer in microwave
    • Denatured samples/ladder for 10 min at 75C before putting on cold rack for 1 min
    • Size selected for brightest band below 200bp
Components 1X Volume 4X Volume
Enzyme digested probes (~12.6ug/400ul) 105 420
TBE-Urea Buffer 2X 105 420
H2O 0 0
Total 210 840
Components 1X Volume 4X Volume
10-bp DNA ladder (1 ug/ul) 1 4
TBE-Urea Buffer 2X 5 20
H2O 4 16
total 10 40

Gel 1:
File:2013-05-29 Gel1SizeSelection 20gap.jpg
Gel 2:
File:2013-05-29 Gel2SizeSelection 20gap.jpg
Gel 3:
File:2013-05-29 Gel3SizeSelection 20gap.jpg
Gel 4:
File:2013-05-29 Gel4SizeSelection 20gap.jpg

  • Centrifuged in 0.5mL tube with punctured holes into a 1.5mL tube
  • Added 450ul TE Buffer and vortexed for 1 hr
  • Had to go to class so Noi finished the rest
  • After precipitation and quantification: 80ul of 14.38ng/ul => total yield = 1.15ug/(193nt*325 Da/nt) = 18.3 pmole

Probe Capture continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-4