Matt:LabNotes/2014-6-27: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
(Created page with "==Agi26k0gap Probe Production Continued== *Continued from: Matt:LabNotes/2014-6-24 ===PAGE Size Selection=== *Run 4 gels of ~3.2ug ssDNA in each **Corresponds to 22.5ul ...")
 
>Mzcai
Line 39: Line 39:
*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.
*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 2.0 mL tube below with pipette tip.
*Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip.
*Added 450 ul of 1X TE buffer
*Added 900 ul of 1X TE buffer
*Vortexed for 45-60 min at 37 C in incubator
*Vortexed for 60 min at 37 C in incubator
*Centrifuged at 15,000 rpm for 3 min at RT
*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 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 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)
*Transfered spnt to fresh 1.5 mL tube
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
*Vortexed and placed the 4 tubes at -80C
*Vortexed and placed the 4 tubes at -80C

Revision as of 21:48, 27 June 2014

Agi26k0gap Probe Production Continued

PAGE Size Selection

  • Run 4 gels of ~3.2ug ssDNA in each
    • Corresponds to 22.5ul
Components 1X Volume 4X Volume
Enzyme digested probes 22.5 90
TBE-Urea Buffer 2X 50 200
Water 27.5 110
Total 100 400
Components 4X Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40
  • Ran at 220V for 30min
  • Cut out brightest band below 200bp

EtOH Precipitation

  • 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 2.0 mL tube below with pipette tip.
  • Added 900 ul of 1X TE buffer
  • Vortexed for 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
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 4 tubes at -80C