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==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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''4X Volume''' |- | Enzyme digested probes||22.5||90 |- | TBE-Urea Buffer 2X||50||200 |- | Water||27.5||110 |- | Total||100||400 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''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 [[File:2014-06-27_Agi26k0gapSizeSelectGel1.jpg]]<br> [[File:2014-06-27_Agi26k0gapSizeSelectGel2.jpg]]<br> [[File:2014-06-27_Agi26k0gapSizeSelectGel3.jpg]]<br> [[File:2014-06-27_Agi26k0gapSizeSelectGel4.jpg]] ===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 8 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 (~450ul per tube) *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2 *Vortexed and placed the 8 tubes at -80C *Spun 8 tubes at 10,000rpm at 4C for 30min *Discard supernatant and add 650ul 75% EtOH and spin at 14,000rpm at 4C for 5min *Discard supernatant and let dry in hood for 10min *Resuspend each tube with 10ul (80ul total) ===TBU Gel Quantification=== *Diluted Agi26k0gap 10X **2ul Agi26k0gap + 8ul H2O + 10ul 2X TBE-Urea buffer *Made DNA Low Mass Ladder with 0.5, 1, and 2 ul {| {{table}} | align="center" style="background:#f0f0f0;"|'''Lane''' | align="center" style="background:#f0f0f0;"|'''1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' | align="center" style="background:#f0f0f0;"|'''4''' | align="center" style="background:#f0f0f0;"|'''5''' | align="center" style="background:#f0f0f0;"|'''6''' | align="center" style="background:#f0f0f0;"|'''7''' |- | Sample||0.5ul DNA Ladder||1ul DNA Ladder||2ul Sample||4ul Sample||6ul Sample||8ul Sample||2ul DNA Ladder |} [[File:2014-06-30_Agi26k0gapTBUGelQuant.jpg]] *[[Media:2014-06-30_Agi26k0gapTBUGelQuantAnalysis.xlsx | Quantification Analysis]] *Agi26k0gap Concentration: 56.9 ng/ul *Total Mass: 56.9 ng/ul * 80ul = 4.55 ug *Total Amount: 4.55ug /(193nt*325 Da/nt) = 72.6 pmole (908nM)
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