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==Agi26k0gap Probe Production== ===Production PCR=== *Make Master Mix for 51 well (7 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)''' |- | [[Matt:LabNotes/2014-5-22#Probe_Expansion_PCR | 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:2014-08-14_Agi26k0gapProductionPCR.JPG|650px]] ===EtOH Precipitation=== *6 15-ml tubes (with 8.5 wells of PCR product each) for 0gap **850ul PCR product **2000ul 100% EtOH **2.7ul GlycoBlue **85ul 3M NaOAc pH 5.2-5.5 *Vortexed and put in -80C for overnight *Centrifuged at 3000rpm at 4C for 30min *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: *~225ul of Agi26k0gap probes: 317.5 ng/ul => 71.4 ug ===Lambda Exonuclease Digestion=== *Divide into seven pcr tubes of 32ul with total amplicon of ~10ug each **Seventh tube had 33ul of amplicon {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Amplicon||32 |- | 10X Lambda Exo Buffer||10 |- | Lambda Exonuclease||10 |- | H2O||48 |- | Total||100 |} *Incubated at 37C for 1hr *Purified with Zymo ssDNA/RNA columns *Eluted with 40ul each *Recombined and measured ssDNA with Nanodrop: ** 89.0ng/ul x 255ul = 22.7ug (63.6% yield) ===Removal of Amplification Adapters=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | ss-amplicon||63.5 |- | USER||5 |- | H2O||11.5 |- | total||80 |} *~5ug ssDNA per tube *Incubated at 37C for 2 hours *Added 15ul of the following {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X DpnII Buffer||10 |- | 100uM RE-DpnII guide oligo||5 |} RE-DpnII guide oligo: 0-gap: RE DpnII V6 *Incubated at 94C for 2 min, then 37C for 3 min *Added 5ul DpnII *Incubated at 37C for 3 hr *Added 100ul of the following {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X DpnII Buffer||10 |- | USER||5 |- | H2O||85 |- | Total||100 |} *Incubated at 37C for overnight (~15 hrs) ===TBU Gel Check=== *Combine the 4 tubes *2ul Sample + 2ul 2X Urea Loading Dye *0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye *Digested sample *Undigested sample: 10nM 1st round amplicon [[File:2014-08-17_Agi26k0gapDigestCheck.jpg]] *Brightest band below 200bp as expected ===Column Purification=== *Used Zymo ssDNA/RNA columns *Total Eluted: 260 ul *Nanodrop quant: 50.8 ng/ul *Yield: 50.8 ng/ul x 260 ul = 13.2 ug ===PAGE Size Selection=== *On 8/26/14 *Run 4 gels of ~3.3ug ssDNA in each **Corresponds to 65ul in each gel {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''4X Volume''' |- | Enzyme digested probes||65||260 |- | TBE-Urea Buffer 2X||65||260 |- | Water||0||0 |- | Total||130||520 |} {| {{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:2014-08-26_Agi26k0gapSizeSelectionGel1.jpg]] [[File:2014-08-26_Agi26k0gapSizeSelectionGel2.jpg]] [[File:2014-08-26_Agi26k0gapSizeSelectionGel3.jpg]] [[File:2014-08-26_Agi26k0gapSizeSelectionGel4.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 for overnight *Spun 8 tubes at 10,000rpm at 4C for 30min *Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min *Discard supernatant and let dry in hood for 10min *Resuspend each tube with 10ul (70ul 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:2014-08-27_Agi26k0gapGelQuant.jpg]] *[[Media:2014-08-27_Agi26k0gapGelQuant.xlsx | Quantification Analysis]] *Agi26k0gap Concentration: 82.4 ng/ul *Total Mass: 82.4 ng/ul * 70ul = 5.77 ug *Total Amount: 5.77 ug /(193nt*325 Da/nt) = 92 pmole (1.314 uM)
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