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== Reaction mix == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1rxn (ul)''' | align="center" style="background:#f0f0f0;"|'''105 rxn mix (ul)''' |- | 10X AmpLigase Buffer||2.50||262.50 |- | DMR330k probe mix (ul)||3.43||360.15 |- | 105nM Oligo suppressor||0.30||31.50 |- | Total volume (ul)||6.23||654.15 |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''sample ID''' | align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 300ng(ul)''' | align="center" style="background:#f0f0f0;"|'''sample ID''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount of bis-cvt gDNA (ng)''' | align="center" style="background:#f0f0f0;"|'''Probe mix (ul)''' | align="center" style="background:#f0f0f0;"|'''10x Amligase buffer (ul)''' | align="center" style="background:#f0f0f0;"|'''1.05uM Oligo suppressor set1&2 (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | A1||29.00||10.34||A1||8.43||300.00||3.43||2.50||0.30||25.00 |- | B1||23.20||12.93||B1||5.84||300.00||3.43||2.50||0.30||25.00 |- | C1||31.60||9.49||C1||9.28||300.00||3.43||2.50||0.30||25.00 |- | D1||33.00||9.09||D1||9.68||300.00||3.43||2.50||0.30||25.00 |- | E1||20.80||14.42||E1||4.35||300.00||3.43||2.50||0.30||25.00 |- | F1||18.98||15.81||F1||2.96||300.00||3.43||2.50||0.30||25.00 |- | G1||28.80||10.42||G1||8.35||300.00||3.43||2.50||0.30||25.00 |- | H1||23.60||12.71||H1||6.06||300.00||3.43||2.50||0.30||25.00 |- | A2||19.34||15.51||A2||3.26||300.00||3.43||2.50||0.30||25.00 |- | B2||25.60||11.72||B2||7.05||300.00||3.43||2.50||0.30||25.00 |- | C2||25.00||12.00||C2||6.77||300.00||3.43||2.50||0.30||25.00 |- | D2||23.20||12.93||D2||5.84||300.00||3.43||2.50||0.30||25.00 |- | E2||23.80||12.61||E2||6.16||300.00||3.43||2.50||0.30||25.00 |- | F2||32.00||9.38||F2||9.40||300.00||3.43||2.50||0.30||25.00 |- | G2||18.92||15.86||G2||2.91||300.00||3.43||2.50||0.30||25.00 |- | H2||20.60||14.56||H2||4.21||300.00||3.43||2.50||0.30||25.00 |- | A3||17.56||17.08||A3||1.69||300.00||3.43||2.50||0.30||25.00 |- | B3||29.00||10.34||B3||8.43||300.00||3.43||2.50||0.30||25.00 |- | C3||29.00||10.34||C3||8.43||300.00||3.43||2.50||0.30||25.00 |- | D3||21.60||13.89||D3||4.88||300.00||3.43||2.50||0.30||25.00 |- | E3||25.80||11.63||E3||7.14||300.00||3.43||2.50||0.30||25.00 |- | F3||23.20||12.93||F3||5.84||300.00||3.43||2.50||0.30||25.00 |- | G3||30.60||9.80||G3||8.97||300.00||3.43||2.50||0.30||25.00 |- | H3||28.00||10.71||H3||8.06||300.00||3.43||2.50||0.30||25.00 |- | A4||22.60||13.27||A4||5.50||300.00||3.43||2.50||0.30||25.00 |- | B4||26.40||11.36||B4||7.41||300.00||3.43||2.50||0.30||25.00 |- | C4||24.00||12.50||C4||6.27||300.00||3.43||2.50||0.30||25.00 |- | D4||28.40||10.56||D4||8.21||300.00||3.43||2.50||0.30||25.00 |- | E4||25.60||11.72||E4||7.05||300.00||3.43||2.50||0.30||25.00 |- | F4||26.40||11.36||F4||7.41||300.00||3.43||2.50||0.30||25.00 |- | G4||23.40||12.82||G4||5.95||300.00||3.43||2.50||0.30||25.00 |- | H4||23.80||12.61||H4||6.16||300.00||3.43||2.50||0.30||25.00 |- | A5||25.20||11.90||A5||6.87||300.00||3.43||2.50||0.30||25.00 |- | B5||24.40||12.30||B5||6.47||300.00||3.43||2.50||0.30||25.00 |- | C5||22.20||13.51||C5||5.26||300.00||3.43||2.50||0.30||25.00 |- | D5||23.80||12.61||D5||6.16||300.00||3.43||2.50||0.30||25.00 |- | E5||25.60||11.72||E5||7.05||300.00||3.43||2.50||0.30||25.00 |- | F5||34.40||8.72||F5||10.05||300.00||3.43||2.50||0.30||25.00 |- | G5||22.40||13.39||G5||5.38||300.00||3.43||2.50||0.30||25.00 |- | H5||14.40||17.00||H5**||1.77||244.80||3.43||2.50||0.30||25.00 |- | A6||26.60||11.28||A6||7.49||300.00||3.43||2.50||0.30||25.00 |- | B6||20.80||14.42||B6||4.35||300.00||3.43||2.50||0.30||25.00 |- | C6||23.60||12.71||C6||6.06||300.00||3.43||2.50||0.30||25.00 |- | D6||20.20||14.85||D6||3.92||300.00||3.43||2.50||0.30||25.00 |- | E6||19.26||15.58||E6||3.19||300.00||3.43||2.50||0.30||25.00 |- | F6||20.80||14.42||F6||4.35||300.00||3.43||2.50||0.30||25.00 |- | G6||27.00||11.11||G6||7.66||300.00||3.43||2.50||0.30||25.00 |- | H6||22.20||13.51||H6||5.26||300.00||3.43||2.50||0.30||25.00 |- | A7||33.80||8.88||A7||9.89||300.00||3.43||2.50||0.30||25.00 |- | B7||23.00||13.04||B7||5.73||300.00||3.43||2.50||0.30||25.00 |- | C7||29.60||10.14||C7||8.63||300.00||3.43||2.50||0.30||25.00 |- | D7||34.20||8.77||D7||10.00||300.00||3.43||2.50||0.30||25.00 |- | E7||33.60||8.93||E7||9.84||300.00||3.43||2.50||0.30||25.00 |- | F7||27.40||10.95||F7||7.82||300.00||3.43||2.50||0.30||25.00 |- | G7||27.80||10.79||G7||7.98||300.00||3.43||2.50||0.30||25.00 |- | H7||24.60||12.20||H7||6.57||300.00||3.43||2.50||0.30||25.00 |- | A8||28.40||10.56||A8||8.21||300.00||3.43||2.50||0.30||25.00 |- | B8||24.60||12.20||B8||6.57||300.00||3.43||2.50||0.30||25.00 |- | C8||31.60||9.49||C8||9.28||300.00||3.43||2.50||0.30||25.00 |- | D8||15.96||17.00||D8**||1.77||271.32||3.43||2.50||0.30||25.00 |- | E8||36.20||8.29||E8||10.48||300.00||3.43||2.50||0.30||25.00 |- | F8||18.00||16.67||F8||2.10||300.00||3.43||2.50||0.30||25.00 |- | G8||33.40||8.98||G8||9.79||300.00||3.43||2.50||0.30||25.00 |- | H8||38.00||7.89||H8||10.88||300.00||3.43||2.50||0.30||25.00 |- | A9||33.40||8.98||A9||9.79||300.00||3.43||2.50||0.30||25.00 |- | B9||40.40||7.43||B9||11.34||300.00||3.43||2.50||0.30||25.00 |- | C9||35.60||8.43||C9||10.34||300.00||3.43||2.50||0.30||25.00 |- | D9||49.80||6.02||D9||12.75||300.00||3.43||2.50||0.30||25.00 |- | E9||23.00||13.04||E9||5.73||300.00||3.43||2.50||0.30||25.00 |- | F9||31.00||9.68||F9||9.09||300.00||3.43||2.50||0.30||25.00 |- | G9||52.80||5.68||G9||13.09||300.00||3.43||2.50||0.30||25.00 |- | H9||25.00||12.00||H9||6.77||300.00||3.43||2.50||0.30||25.00 |- | A10||29.60||10.14||A10||8.63||300.00||3.43||2.50||0.30||25.00 |- | B10||33.20||9.04||B10||9.73||300.00||3.43||2.50||0.30||25.00 |- | C10||35.60||8.43||C10||10.34||300.00||3.43||2.50||0.30||25.00 |- | D10||37.40||8.02||D10||10.75||300.00||3.43||2.50||0.30||25.00 |- | E10||31.00||9.68||E10||9.09||300.00||3.43||2.50||0.30||25.00 |- | F10||17.40||17.00||F10**||1.77||295.80||3.43||2.50||0.30||25.00 |- | G10||38.80||7.73||G10||11.04||300.00||3.43||2.50||0.30||25.00 |- | H10||33.00||9.09||H10||9.68||300.00||3.43||2.50||0.30||25.00 |- | A11||40.60||7.39||A11||11.38||300.00||3.43||2.50||0.30||25.00 |- | B11||33.60||8.93||B11||9.84||300.00||3.43||2.50||0.30||25.00 |- | C11||14.32||17.00||C11**||1.77||243.44||3.43||2.50||0.30||25.00 |- | D11||33.00||9.09||D11||9.68||300.00||3.43||2.50||0.30||25.00 |- | E11||29.40||10.20||E11||8.57||300.00||3.43||2.50||0.30||25.00 |- | F11||24.60||12.20||F11||6.57||300.00||3.43||2.50||0.30||25.00 |- | G11||22.20||13.51||G11||5.26||300.00||3.43||2.50||0.30||25.00 |- | H11||36.80||8.15||H11||10.62||300.00||3.43||2.50||0.30||25.00 |- | A12||27.20||11.03||A12||7.74||300.00||3.43||2.50||0.30||25.00 |- | B12||33.80||8.88||B12||9.89||300.00||3.43||2.50||0.30||25.00 |- | C12||27.00||11.11||C12||7.66||300.00||3.43||2.50||0.30||25.00 |- | D12||35.40||8.47||D12||10.30||300.00||3.43||2.50||0.30||25.00 |- | E12||35.40||8.47||E12||10.30||300.00||3.43||2.50||0.30||25.00 |- | F12||26.40||11.36||F12||7.41||300.00||3.43||2.50||0.30||25.00 |- | G12||26.00||11.54||G12||7.23||300.00||3.43||2.50||0.30||25.00 |- | H12||46.00||6.52||H12||12.25||300.00||3.43||2.50||0.30||25.00 |}<br> * Add H2O to the 96-well plate first then add bis-cvt gDNA. Note: Try to deliver reagents to the bottom of the well * Prepare reaction mix in 1.5 mL tube by pipetting probe, oligo suppressor and 10x AmLigase buffer and mix well. * Aliquot reaction mix in striptube (8 tubes): 81.77ul * Aliquot reaction mix 6.23ul with multichannel pipette and mix well * Put mineral oil in basin ~3 mL * Layer the capture reaction with 25ul mineral oil with multichannel pipette. * Seal the plate with microseal and make sure that every well is completely sealed to prevent evaporation * Check the level of every well that they have the same level * Centrifuge the capture plate for 3 min and place the plate on PCR machine with the following profile, '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2.5ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (2011_08_17) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_08_18) === SLN mix === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Stock conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Prepare volume 275ul''' |- | AmpliTaq Stoffel||10.00||U/ul||2.00||U/ul||55.00 |- | AmpLigase||5.00||U/ul||0.50||U/ul||27.50 |- | dNTP||1000.00||uM||100.00||uM||27.50 |- | 10x AmpLigase Buffer||10.00||x||1.00||x||27.50 |- | H2O||||||||||137.50 |- | Total||||||||||275.00 |} * Aliquot 34.38 ul to stripp tube * Add 2.5ul of SLN mix to each well with multichannel pipettes === ExoI/III preparation === * Mix 110 ul each of ExoI and ExoIII and aliquot 27.5ul into striptube * Add 2ul to each well with multichannel pipettes
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