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===Procedure=== Sample <br> {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''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 <br> {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''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: <br> [[File:2014-03-05_Agi26k0gapPrep_Gel1.jpg| 450px]] Gel 2: <br> [[File:2014-03-05_Agi26k0gapPrep_Gel2.jpg| 450px]] Gel 3: <br> [[File:2014-03-05_Agi26k0gapPrep_Gel3.jpg| 450px]] *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 ====Day 2==== '''Ethanol Precipitation after PAGE size selection''' *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 (60ul total) '''TBU Gel Quantification Plan''' *Dilute 2ul into 20ul *Mix 1:1 with 2X buffer (20X dilution) *Load sample wells with 2, 4, 6, 8, and 10 ul *Load Low Mass DNA ladders in 0.5, 1, and 2 ul {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''0.5 ladder''' | align="center" style="background:#f0f0f0;"|'''1 ladder''' | align="center" style="background:#f0f0f0;"|''' 2 ul ''' | align="center" style="background:#f0f0f0;"|''' 4 ul ''' | align="center" style="background:#f0f0f0;"|''' 6 ul ''' | align="center" style="background:#f0f0f0;"|''' 8 ul ''' | align="center" style="background:#f0f0f0;"|''' 10 ul ''' | align="center" style="background:#f0f0f0;"|'''2 ladder''' |- | DNA volume||0.5||1||0.1||0.2||0.3||0.4||0.5||2 |- | H2O||1.5||1||0.9||1.8||2.7||3.6||4.5||0 |- | 2X Buffer||2||2||1||2||3||4||5||2 |- | Total||4||4||2||4||6||8||10||4 |- | |} [[File:2014-03-07_Quantify_Agi26k0gap.jpg|650px]] *[[Media:Agi26k0gap_GelQuantification.xlsx|Intensity to [DNA] calculations]] *20X diluated sample concentration is 1.64ng/ul *Starting sample concentration: 32.8ng/ul -> total yield about 1.9ug/(193nt*325 Da/nt) = 30.3 pmole
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