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