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= Test BSPP DNA capture with DMR330k probe set with all new reagents = * '''Sample:''' * 3 human iPS lines with single Nonsynonymous mutations and 2 GA samples from Kang Zhang's lab ** 2HFF 8d-10 (conc. 214.6 ng/ul) ** 218HFF 2-11 (conc. 411.1 ng/ul) ** 218 HFF 4-11 (conc. 359.6 ng/ul) ** GA sample well A5 (conc. of bis-cvt DNA 88 ng/ul) ** GA sample well ID B5 (conc. of bis-cvt DNA 50.4 ng/ul) Note: Since S5 probe was lately made and I a little bit concern about the stuffs in the probe like salts or acrylamide contaminations even it never show the effect before (The volume of S5 probe is the largest in the reaction). I will set up the reaction with or without S5 probe. {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Sample ID in Zhang lab''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''volume for 1200ng (ul)''' | align="center" style="background:#f0f0f0;"|'''H20 (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount for bis-cvt (ng)''' | align="center" style="background:#f0f0f0;"|'''CT conversion reagent (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | 2HFF 8d_10||8d_10||214.60||5.59||14.41||1200.00||130.00||150.00 |- | 218HHF211||2_11||411.10||2.92||17.08||1200.00||130.00||150.00 |- | 218HHF411||4_11||359.60||3.34||16.66||1200.00||130.00||150.00 |} * Note: prepared two reactions for each samples<br> == Qubit ss-DNA assay == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Conc. In Qubit (ng/mL)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Final conc (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 250ng''' |- | 8d_10||95.60||38.00||19.12||726.56||13.08 |- | 2_11||69.80||38.00||13.96||530.48||17.91 |- | 4_11||79.30||38.00||15.86||602.68||15.76 |}<br> = 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> = PAGE quantification and MmeI digestion set up = {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Conc.(ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume(ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' |- | 8d_10||10.88||50.00||543.78 |- | 2_11||11.75||50.00||587.57 |- | 4_11||9.44||50.00||471.83 |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Conc.(ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume(ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''10X NEB buffer4''' | align="center" style="background:#f0f0f0;"|'''5mM SAM''' | align="center" style="background:#f0f0f0;"|'''MmeI''' | align="center" style="background:#f0f0f0;"|'''Total volume(ul)''' |- | 8d_10||10.88||22.99||250.00||7.21||4.00||0.80||5.00||40.00 |- | 2_11||11.75||21.27||250.00||8.93||4.00||0.80||5.00||40.00 |- | 4_11||9.44||26.49||250.00||3.71||4.00||0.80||5.00||40.00 |}<br> * Prepare reaction mix<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''3.2 rxn''' |- | 10x NEB buffer4||4.00||12.80 |- | 5mM SAM||0.80||2.56 |- | MmeI||5.00||16.00 |- | Total||9.80||Aliquot 9.8 ul |}<br> * Prepare 6.4ul of 5mM SAM by adding 1ul of 32mM SAM to 5.4ul H2O<br> * Incubate 37C 2hr * purify with 1 vol. Ampure beads, elute with 25 ul EB * PAGE quantify DNA concentration (loaded sample 2ul) [[File:ZhangLab_2 2011-08-11 08hr 41min_MmeI.jpg| 300px]]<br> = N2 adaptor ligation set up = == Adaptor preparation == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Final conc. (uM)''' |- | 100uM PE_N2_adaptor ||2.00||20.00 |- | 100uM PE_b_A ||2.00||20.00 |- | 10x Stoffel buffer ||1.00||1x |- | H2O ||5.00|| |- | Total||10.00|| |}<br> '''Program''' 94C 2min -> 0.2C/sec to 20C -> 4C hold<br> Adapters to ligated product ratio: 10:1<br> Est length of digested products: 212bp (after MmeI)<br> MW digested products = (212bp*607.4 D/bp +157.9 D) = 128.927kD = 128,927 g/mole<br> For 100 ng digested product = 100ng / 128,927g/mole = 7.76E-4 nmole * 10:1 = 0.008 nmole adapters required.<br> 20uM Adapters (ul) = 0.008nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.008nmoles/ (20E-3 nmoles/ul) = 0.40 ul <br> * 0.40 ul of 20uM adapters per 100ng digested products. * so 0.24ul of 20uM adapters per 60ng digested products. {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''conc.(ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' | align="center" style="background:#f0f0f0;"|'''N2 adapters conc (uM)''' | align="center" style="background:#f0f0f0;"|'''N2 adaptors volume (ul)''' | align="center" style="background:#f0f0f0;"|'''5xQuickLigase buffer''' | align="center" style="background:#f0f0f0;"|'''QuickLigase (ul)''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''Total (ul)''' |- | 8d_10||3.19||18.80||60.00||20uM||0.24||8.00||1.00||11.96||40.00 |- | 2_11||3.99||15.02||60.00||20uM||0.24||8.00||1.00||15.74||40.00 |- | 4_11||2.78||21.57||60.00||20uM||0.24||8.00||1.00||9.19||40.00 |}<br> ''' Reaction mix''' {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Reagents''' | align="center" style="background:#f0f0f0;"|'''1x''' | align="center" style="background:#f0f0f0;"|'''4.5 rxn mix''' |- | 5xQuickLigase buffer||8.00||36.00 |- | QuickLigase||1.00||4.50 |- | 20uM N2 adapters||0.24||1.08 |- | Total||9.24||Aliquot 9.24ul |}>br> * Incubate at RT for 15 min * Purify with Ampure beads, elute with 30ul EB == Amplification with N2 barcoded primers == * From the previous experiment and optimization, Use 2% N2 ligated DNA template in PCR {| {{table}} | align="center" style="background:#f0f0f0;"|'''Reagents''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''7.2 rxn mix''' |- | N2 adapter ligated template||2.00||0.00 |- | 10uM PCR_F||1.00||7.20 |- | 10uM PCR_R.N2 IndX(X= 21,2 2 & 23)||1.00||0.00 |- | 50x SYBG||0.40||2.88 |- | 2x PhusionHF MM||50.00||360.00 |- | H2O||45.60||328.32 |- | Total volume (ul)||100.00||aliquot 97ul |}<br> * Add 1ul of PCR_R.N2 IndX and 2ul DNA template {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''IndX''' |- | 8d_10||PCR_R.N2Ind21 |- | 2_11||PCR_R.N2Ind22 |- | 4_11||PCR_R.N2Ind23 |}<br> '''Program''' 98C 30sec (98C 10sec -> 63C 20sec-> 72C 30sec) x 12 cycles 72C 2min 15C hold<br> * Purify amplicon with 0.8 volume Ampure beads, elute with 50ul EB each tube (total = 100ul) * PAGE quantify DNA concentration (loaded sample 1ul)<br> [[File:ZhangLab_2 2011-08-11 11hr 57min_N2Amp.jpg| 450px]]<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Yields''' | align="center" style="background:#f0f0f0;"|'''Volume for 200ng (ul)''' |- | 8d_10||2.75||100.00||275.23||72.67 |- | 2_11||3.05||100.00||305.29||65.51 |- | 4_11||2.75||100.00||274.65||72.82 |}<br> * Perform PAGE size selection with 6% TBE 5-well gel (1gel) * Total volume of of multiplexed libraries: 24ul [[File:ZhangLab_2 2011-08-11 15hr 21min_NP3smuiPSSeqLib.jpg| 250px]]<br> * 0.6 and 0.3ul of sequencing libraries were analyzed by PAGE quantification, conc.: 10.58ng/ul, 51.8nM (336bp), yields = 10.58 ng/ul * 24ul = 253.9ng * Qubit quantification: ng/ul * Sequencing: '''Sample ID:NP-3SMUips-Ind21_23-Aug42011, HL100 run 110811 SE 80+7bp, s1-s3'''
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