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==Agi26k0gap Probe Production== *Reference: [[Matt:LabNotes/2013-5-10]] *Use 20nM aliquot of Agilent oligos in tube labeled "FISHYB V1 probes 5/07/13" ===Probe Expansion PCR=== *Expansion PCR in 200ul total: 2 tubes of 100ul for 0bp gap probes with AP1V6 primers {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''0 gap probes (ul)''' |- | Agi26k (20nM)||10 |- | 2x Kapa SYBG qPCR MM||100 |- | 100uM pAP1V6U||0.8 |- | 100uM AP2V6||0.8 |- | H2O||88.4 |- | Total||200 |- | |} *Mixed thoroughly and then split into two tubes 94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 12 -> 72C 3min -> 15C hold [[File:20140522_Agi26k0gapExpansionPCR.JPG| 450px]] *Purified with 2 Qiagen columns *Eluted with 40ul each tube and combined into two tubes (80ul total) *Nanodrop: 32.2 ng/ul -> 32.2ng/ul / (228bp*660Da/bp) = 214nM *Dilute 2.34ul in 47.66ul H2O *Final volumes: **75ul of 214nM **50ul of 10nM ===Production PCR=== *Make Master Mix for 46 well (6 strips) *0 gap first round amplicon -> V6 primers {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn (ul)''' | align="center" style="background:#f0f0f0;"|'''50x rxn (ul)''' |- | First round amplicon (10nM)||0.2||10 |- | 2x Kapa SYBR MM||50||2500 |- | 100uM pAP1V6U||0.4||20 |- | 100uM AP2V6||0.4||20 |- | H2O||49||2450 |- | Total||100||5000 |- | |} 98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold [[File:052314_Agi26k0gapProductionPCR.JPG |650px]] ===EtOH Precipitation=== *6 5-ml tubes (with 8 wells of PCR product each) for 0gap **800ul PCR product **2000ul 100% EtOH **2.7ul GlycoBlue **80ul 3M NaOAc pH 5.2-5.5 *Vortexed and put in -80C for overnight *Centrifuged at 3000rpm at 4C for 30 min *Discarded supernatant and added 800ul of cold 80% EtOH *Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes *Centrifuged 1.5ml tubes at 15,000rpm for 5 min on table-top centrifuge *Discarded supernatant and air-dried for 5 min in hood *Resuspended DNA with 100ul H2O ===Qia Column Purification=== *Purified 6 tubes of 100ul in 6 columns following Qiagen protocol *Eluted each column with 40ul and combined all probes into one 1.5ml *Measured concentration with Nanodrop: ~220ul of Agi26k0gap probes: 295.8 ng/ul => 65.1ug ===Lambda Exonuclease Digestion=== *Divide each tube of 220ul into two 1.5ml tubes of 110ul with total amplicon of ~30ug {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' |- | Amplicon||110 |- | 10X Lambda Exo Buffer||15 |- | Lambda Exonuclease||10 |- | H2O||15 |- | total||150 |- | |} *Incubated at 37C for 1hr *Purified each of the tubes with 3 columns (6 columns total) of Zymo ssDNA/RNA kit *Eluted with 27ul each *Recombined and measured ssDNA with Nanodrop: **49.2ng/ul x 160ul = 7.87ug (12% yield) ***Why is it so low? Expected to be about 40%... **Low yield could be because Zymo kit is for ssDNA between 17nt-200nt and these oligos are slightly above 200nt before USER & DpnII digestion *Noi suggests I may have had too much DNA in each tube for lambda digestion. She says 10ug DNA for 100ul reaction (15ug/150ul reaction) <!-- To continue see: [[Matt:LabNotes/2013-5-13]] --> *Continued on: [[Matt:LabNotes/2014-6-24]]
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