Matt:LabNotes/2014-3-5

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Revision as of 21:05, 5 March 2014 by >Mzcai (Created page with "==Agi26k0gap Padlock Probe Prep== *Taking column purified 0gap probes from Matt:LabNotes/2013-5-16 *Run 3 gels each with 60ul ===Procedure=== Sample <br> {| {{table}} | ...")
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Agi26k0gap Padlock Probe Prep

Procedure

Sample

Components 1X Volume 3X Volume
Enzyme digested probes (~10ug/180ul) 58 174
TBE-Urea Buffer 2X 58 174
Water 0 0
Total 116 348

3 Low Mass DNA Ladders

Components 3X Volume
Low Mass DNA ladder (1 ug/ul) 3
TBE-Urea Buffer 2X 15
H2O 12
Total 30
  • Ran the 3 gels at 220V for 30min
  • Shake in SYBR Gold for 6+ minutes
  • Cut out band below 200bp

Gel 1:
File:2014-03-05 Agi26k0gapPrep Gel1.jpg

Gel 2:
File:2014-03-05 Agi26k0gapPrep Gel2.jpg

Gel 3:
File:2014-03-05 Agi26k0gapPrep Gel3.jpg

  • Put cut out gel in 0.5mL tube (with hole 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 3.5hr 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