Matt:LabNotes/2014-6-23: Difference between revisions

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(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: Ladder, Ran Hex, Poly(dT), FISSEQ_RT
*Gel 1:
*Gel 2: Top 48, No Enzyme, Nextera +, Ladder
[[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.
#Selected size: 200-800bp but went a little conservative so more like 250bp-750bp
*Transfered the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
#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.
*Added 450 ul of 1X TE buffer
#Transfered the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
*Vortexed for 45-60 min at 37 C in incubator
#Added 450 ul of 1X TE buffer
*Centrifuged at 15,000 rpm for 3 min at RT
#Vortexed for 45-60 min at 37 C in incubator
*Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min
#Centrifuged at 15,000 rpm for 3 min at RT
*Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
#Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min
*Transfered spnt to fresh 1.5 mL tube (volume ~ 400ul/ tube)
#Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
#Transfered spnt to fresh 1.5 mL tube (volume ~ 400ul/ tube)
*Vortexed and placed the 6 tubes at -80C overnight
#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

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

  1. Selected size: 200-800bp but went a little conservative so more like 250bp-750bp
  2. 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.
  3. Transfered the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
  4. Added 450 ul of 1X TE buffer
  5. Vortexed for 45-60 min at 37 C in incubator
  6. Centrifuged at 15,000 rpm for 3 min at RT
  7. Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min
  8. Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
  9. Transfered spnt to fresh 1.5 mL tube (volume ~ 400ul/ tube)
  10. Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
  11. Vortexed and placed the 6 tubes at -80C for 20 minutes
  12. Spun 6 tubes in -80C at 10,000rpm at 4C for 30min
  13. Discard supernatant and add 650ul 75% EtOH to wash and spin at 14,000rpm at 4C for 5 min
  14. Discard supernatant and let dry in hood 10 min
  15. Resuspended each of the 6 tubes with 10ul H2O