Matt:LabNotes/2014-6-23: Difference between revisions
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>Mzcai (Created page with "===Size Selection=== *Ladder: 0.5ul Low Mass Ladder + 2.5ul H2O + 2ul Loading Dye *Samples: 5ul Sample + 2ul Loading Dye *Gel 1: Ladder, Ran Hex, Poly(dT), FISSEQ_RT *Gel 2: ...") |
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===Size Selection=== | *Continued from: [[Matt:LabNotes/2014-6-18]] | ||
===Gel Size Selection=== | |||
*Ladder: 0.5ul Low Mass Ladder + 2.5ul H2O + 2ul Loading Dye | *Ladder: 0.5ul Low Mass Ladder + 2.5ul H2O + 2ul Loading Dye | ||
*Samples: 5ul Sample + 2ul Loading Dye | *Samples: 5ul Sample + 2ul Loading Dye | ||
*Gel 1: | *Gel 1: | ||
*Gel 2: | [[File:2014-06-24_SizeSelection_Gel1.jpg | 450px]] | ||
*Gel 2: | |||
[[File:2014-06-24_SizeSelection_Gel2.jpg | 450px]] | |||
#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 |
Latest revision as of 20:54, 25 June 2014
- 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