Matt:LabNotes/2014-6-23: Difference between revisions
Jump to navigation
Jump to search
>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: ...") |
>Mzcai mNo edit summary |
||
Line 1: | Line 1: | ||
*Continued from: [[Matt:LabNotes/2014-6-18]] | |||
===Size Selection=== | ===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]] | |||
#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 |
Revision as of 01:11, 25 June 2014
- Continued from: Matt:LabNotes/2014-6-18
Size Selection
- 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
- 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