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