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= Capture set up = === Probe preparation and reaction mix === == Include S5 probe == * 5 samples {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Subset ID''' | align="center" style="background:#f0f0f0;"|'''Adjusted Multiplier based on qPCR (Multiplier E)''' | align="center" style="background:#f0f0f0;"|'''Actual amount of probe required (probe:target ratio, 100:1)''' | align="center" style="background:#f0f0f0;"|'''Multiplier E x amount probe required''' | align="center" style="background:#f0f0f0;"|'''Probe concentration (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 6rxn (ul)''' |- | DMR.s1||0.05||2.24||0.11||0.39||0.29||1.72 |- | DMR.s2||0.07||2.24||0.16||0.35||0.47||2.79 |- | DMR.s3||0.23||18.90||4.41||23.20||0.19||1.14 |- | DMR.s4||1.00||18.90||18.90||23.20||0.81||4.89 |- | DMR.s5**||4.83||18.90||91.29||29.20||3.13||18.76 |- | DMR.Exp1||0.23||18.90||4.44||31.88||0.14||0.83 |- | DMR.Exp2||0.19||18.90||3.50||24.11||0.15||0.87 |- | DMR.Exp3||0.27||18.90||5.03||28.90||0.17||1.04 |- | 1.05uM Oligo suppressor-set1||||||||||0.13||0.75 |- | 1.05uM Oligo suppressor-set2||||||||||0.13||0.75 |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1rxn (ul)''' | align="center" style="background:#f0f0f0;"|'''6 rxn mix (ul)''' |- | 10X AmpLigase Buffer||2.50||15.00 |- | DMR330k probe mix (ul)||5.34||32.04 |- | 105nM Oligo suppressor||0.25||1.50 |- | Total volume (ul)||8.09||48.54 |}<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 250ng(ul)''' | 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)''' |- | 8d_10||19.12||13.08||3.83||250.00||5.34||2.50||0.25||25.00 |- | 2_11||13.96||17.91||-1.00||250.00||5.34||2.50||0.25||25.00 |- | 4_11||15.86||15.76||1.15||250.00||5.34||2.50||0.25||25.00 |- | A5 (GA)||88.00||2.84||14.07||250.00||5.34||2.50||0.25||25.00 |- | B5 (GA)||50.40||4.96||11.95||250.00||5.34||2.50||0.25||25.00 |}<br> == Not include S5 probe == * 3 samples {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Subset ID''' | align="center" style="background:#f0f0f0;"|'''Adjusted Multiplier based on qPCR (Multiplier E)''' | align="center" style="background:#f0f0f0;"|'''Actual amount of probe required (probe:target ratio, 100:1)''' | align="center" style="background:#f0f0f0;"|'''Multiplier E x amount probe required''' | align="center" style="background:#f0f0f0;"|'''Probe concentration (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 4rxn (ul)''' |- | DMR.s1||0.05||2.24||0.11||0.39||0.29||1.15 |- | DMR.s2||0.07||2.24||0.16||0.35||0.47||1.86 |- | DMR.s3||0.23||18.90||4.41||23.20||0.19||0.76 |- | DMR.s4||1.00||18.90||18.90||23.20||0.81||3.26 |- | DMR.Exp1||0.23||18.90||4.44||31.88||0.14||0.56 |- | DMR.Exp2||0.19||18.90||3.50||24.11||0.15||0.58 |- | DMR.Exp3||0.27||18.90||5.03||28.90||0.17||0.70 |- | 1.05uM Oligo suppressor-set1||||||||||0.13||0.50 |- | 1.05uM Oligo suppressor-set2||||||||||0.13||0.50 |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1rxn (ul)''' | align="center" style="background:#f0f0f0;"|'''4 rxn mix (ul)''' |- | 10X AmpLigase Buffer||2.50||10.00 |- | DMR330k probe mix (ul)||2.22||8.88 |- | 105nM Oligo suppressor||0.25||1.00 |- | Total volume (ul)||4.97||19.88 |}<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 250ng(ul)''' | 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)''' |- | 8d_10||19.12||13.08||6.95||250.00||2.22||2.50||0.25||25.00 |- | 2_11||13.96||17.91||2.12||250.00||2.22||2.50||0.25||25.00 |- | 4_11||15.86||15.76||4.27||250.00||2.22||2.50||0.25||25.00 |}<br> '''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_05) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_08_06) === 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 23 ul''' |- | AmpliTaq Stoffel||10.00||U/ul||2.00||U/ul||4.60 |- | AmpLigase||5.00||U/ul||0.50||U/ul||2.30 |- | dNTP||1000.00||uM||100.00||uM||2.30 |- | 10x AmpLigase Buffer||10.00||x||1.00||x||2.30 |- | H2O||||||||||11.50 |}<br> == Amplification == * Performed PCR to test if the capture work well in 50ul reaction {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Reagents''' | align="center" style="background:#f0f0f0;"|'''volume (ul)''' | align="center" style="background:#f0f0f0;"|'''8.5 rxn mix''' |- | captured template||4.00||0.00 |- | 100uM AmpFV6.3NH2||0.10||0.85 |- | 100uM AmpRV6.3NH2||0.10||0.85 |- | 50x SYBG||0.40||3.40 |- | 2x PhusionHF MM||25.00||212.50 |- | H2O||20.40||173.40 |- | Total volume (ul)||50.00||aliquot 46ul add 4ul template |}<br> '''Program''' 98C 30sec<br> (98C 10sec -> 58C 20sec-> 72C 20sec) x 20 cycles <br> 72C 2min<br> 15C hold. 2011_08_10 * Perform PCR to amplify the three human iPS line samples {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Reagents''' | align="center" style="background:#f0f0f0;"|'''volume (ul)''' | align="center" style="background:#f0f0f0;"|'''6.5 rxn mix''' |- | captured template||8.00||0.00 |- | 100uM AmpFV6.3NH2||0.20||1.30 |- | 100uM AmpRV6.3NH2||0.20||1.30 |- | 50x SYBG||0.80||5.20 |- | 2x PhusionHF MM||50.00||325.00 |- | H2O||40.80||265.20 |- | Total volume (ul)||100.00||aliquot 92ul add 8ul template |}<br> '''Program''' 98C 30sec<br> (98C 10sec -> 58C 20sec-> 72C 20sec) x 20 cycles <br> 72C 2min<br> 15C hold. * Purify with 0.8 vol. Ampure beads, elute with 25ul EB/tube, so total volume = 50ul * PAGE quantify DNA concentration (loaded sample 0.5 ul) [[File:ZhangLab_2 2011-08-10 19hr 02min_purified amplicon.jpg| 300px]]<br> == Ct values == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Ave Ct value''' |- | 8d_10||15.08 |- | 2_11||14.59 |- | 4_11||15.77 |}<br>
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