Matt:LabNotes/2014-6-23
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- Continued from: Matt:LabNotes/2014-6-18
Gel Size Selection[edit]
- Ladder: 0.5ul Low Mass Ladder + 2.5ul H2O + 2ul Loading Dye
- Samples: 5ul Sample + 2ul Loading Dye
- Gel 1:
File:2014-06-24 SizeSelection Gel1.jpg
- Gel 2:
File:2014-06-24 SizeSelection Gel2.jpg
- Selected size: 200-800bp but went a little conservative so more like 250bp-750bp
- 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 for 20 minutes
- 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