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= MONOD V1N3 BSPP capture (repeat sensitivity test) = '''Link to calendar: [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014]]''' * I repeated MONOD V1N3 capture experiment on 3014-05-29: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-5-29]] * I reduced capture volume to 10ul. * I reduced incubation time after adding SLN mix between 4-6h since I think that 20h is not necessary for polymerization and may cause more back ground if DNA template level is too low. * I will add experimental procedures later == Bisulfite conversion == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Mix''' | align="center" style="background:#f0f0f0;"|'''82.1ng/ul JK DNA''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Aliquot''' |- | 25ng||1.28||82.72||20.00 |- | 50ng||2.56||81.44||20.00 |- | 100ng||5.12||78.88||20.00 |- | 200ng||10.23||73.77||20.00 |} {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Amount (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:60px;background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="width:60px;background:#f0f0f0;"|'''Lightnig Conversion reagent''' | align="center" style="width:60px;background:#f0f0f0;"|'''Total''' |- | JK-1||82.1||25||0.30||19.70||130||150.00 |- | JK-2||82.1||25||0.30||19.70||130||150.00 |- | JK-3||82.1||25||0.30||19.70||130||150.00 |- | JK-4||82.1||25||0.30||19.70||130||150.00 |- | JK-5||82.1||50||0.61||19.39||130||150.00 |- | JK-6||82.1||50||0.61||19.39||130||150.00 |- | JK-7||82.1||50||0.61||19.39||130||150.00 |- | JK-8||82.1||50||0.61||19.39||130||150.00 |- | JK-9||82.1||100||1.22||18.78||130||150.00 |- | JK-10||82.1||100||1.22||18.78||130||150.00 |- | JK-11||82.1||100||1.22||18.78||130||150.00 |- | JK-12||82.1||100||1.22||18.78||130||150.00 |- | JK-13||82.1||200||2.44||17.56||130||150.00 |- | JK-14||82.1||200||2.44||17.56||130||150.00 |- | JK-15||82.1||200||2.44||17.56||130||150.00 |- | JK-16||82.1||200||2.44||17.56||130||150.00 |} * Elute with 10ul Elution buffer * Use 1ul for Qubit ssDNA assay ==== Qubit ssDNA assay results ==== * I did many bisulfite conversion reactions to compare the recovery rate with different inputs {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:60px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:60px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:100px;background:#f0f0f0;"|'''Sample conc. (ng/ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Yield in 9ul (ng)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Input DNA (ng)''' | align="center" style="width:100px;background:#f0f0f0;"|'''% Recovery''' |- | JK-1||3.8||ng/mL||1||200||0.760||6.84||bgcolor="lightcyan"|25||bgcolor="lightcyan"|27.36 |- | JK-2||4.9||ng/mL||1||200||0.980||8.82||bgcolor="lightcyan"|25||bgcolor="lightcyan"|35.28 |- | JK-3||2.5||ng/mL||1||200||0.500||4.50||bgcolor="lightcyan"|25||bgcolor="lightcyan"|18.00 |- | JK-4||5.11||ng/mL||1||200||1.022||9.20||bgcolor="lightcyan"|25||bgcolor="lightcyan"|36.79 |- | JK-5||10.8||ng/mL||1||200||2.160||19.44||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|38.88 |- | JK-6||9.06||ng/mL||1||200||1.812||16.31||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|32.62 |- | JK-7||12.3||ng/mL||1||200||2.460||22.14||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|44.28 |- | JK-8||11.2||ng/mL||1||200||2.240||20.16||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|40.32 |- | JK-9||25||ng/mL||1||200||5.000||45.00||bgcolor="aqua"|100||bgcolor="aqua"|45.00 |- | JK-10||26.7||ng/mL||1||200||5.340||48.06||bgcolor="aqua"|100||bgcolor="aqua"|48.06 |- | JK-11||28.4||ng/mL||1||200||5.680||51.12||bgcolor="aqua"|100||bgcolor="aqua"|51.12 |- | JK-12||24.3||ng/mL||1||200||4.860||43.74||bgcolor="aqua"|100||bgcolor="aqua"|43.74 |- | JK-13||90.1||ng/mL||1||200||18.020||162.18||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|81.09 |- | JK-14||89||ng/mL||1||200||17.800||160.20||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|80.10 |- | JK-15||79.7||ng/mL||1||200||15.940||143.46||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|71.73 |- | JK-16||85.4||ng/mL||1||200||17.080||153.72||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|76.86 |} == BSPP capture set up == {| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''strip 1''' | align="center" style="width:60px;background:#f0f0f0;"|'''5:500''' | align="center" style="width:60px;background:#f0f0f0;"|'''5:500''' | align="center" style="width:60px;background:#f0f0f0;"|'''10:500''' | align="center" style="width:60px;background:#f0f0f0;"|'''10:500''' | align="center" style="width:60px;background:#f0f0f0;"|'''25:500''' | align="center" style="width:60px;background:#f0f0f0;"|'''25:500''' | align="center" style="width:60px;background:#f0f0f0;"|'''50:500''' | align="center" style="width:60px;background:#f0f0f0;"|'''50:500''' |- | BIS-DNA required (ng)||5||5||10||10||25||25||50||50 |- | JK #||JK-1||JK-2||JK-5||JK-6||JK-9||JK-10||JK-13||JK-14 |- | Conc.||0.760||0.980||2.160||1.812||5.000||5.340||18.020||17.800 |- | Volume (ul)||6.58||5.10||4.63||5.52||5.00||4.68||2.77||2.81 |- | H2O (ul)||0.27||1.75||2.07||1.18||1.25||1.57||2.72||2.69 |- | V1N3 amount (ng)||0.11||0.11||0.22||0.22||0.55||0.55||1.10||1.10 |- | V1N3 conc. (ng/ul)||0.73||0.73||0.73||0.73||0.73||0.73||0.73||0.73 |- | V1N3 volume (ul)||0.15||0.15||0.30||0.30||0.75||0.75||1.50||1.50 |- | 10X Ampligase Buffer||1.00||1.00||1.00||1.00||1.00||1.00||1.00||1.00 |- | H2O in Mix||2.00||2.00||2.00||2.00||2.00||2.00||2.00||2.00 |- | Total||10.00||10.00||10.00||10.00||10.00||10.00||10.00||10.00 |} <br> {| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''strip 2''' | align="center" style="width:60px;background:#f0f0f0;"|'''5:1000''' | align="center" style="width:60px;background:#f0f0f0;"|'''5:1000''' | align="center" style="width:60px;background:#f0f0f0;"|'''10:1000''' | align="center" style="width:60px;background:#f0f0f0;"|'''10:1000''' | align="center" style="width:60px;background:#f0f0f0;"|'''25:1000''' | align="center" style="width:60px;background:#f0f0f0;"|'''25:1000''' | align="center" style="width:60px;background:#f0f0f0;"|'''50:1000''' | align="center" style="width:60px;background:#f0f0f0;"|'''50:1000''' |- | BIS-DNA required (ng)||5||5||10||10||25||25||50||50 |- | JK #||JK-4||JK-5||JK-7||JK-8||JK-11||JK-12||JK-15||JK-16 |- | Conc.||1.022||2.160||2.460||2.240||5.680||4.860||15.940||17.080 |- | Volume (ul)||4.89||2.31||4.07||4.46||4.40||5.14||3.14||2.93 |- | H2O (ul)||1.81||4.38||2.33||1.93||1.09||0.35||0.85||1.06 |- | V1N3 amount (ng)||0.22||0.22||0.44||0.44||1.10||1.10||2.20||2.20 |- | V1N3 conc. (ng/ul)||0.73||0.73||0.73||0.73||0.73||0.73||0.73||0.73 |- | V1N3 volume (ul)||0.30||0.30||0.60||0.60||1.50||1.50||3.01||3.01 |- | 10X Ampligase Buffer||1.00||1.00||1.00||1.00||1.00||1.00||1.00||1.00 |- | H2O in Mix||2.00||2.00||2.00||2.00||2.00||2.00||2.00||2.00 |- | Total||10.00||10.00||10.00||10.00||10.00||10.00||10.00||10.00 |} <br> {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''strip 3''' | align="center" style="width:60px;background:#f0f0f0;"|'''NTC:500''' | align="center" style="width:60px;background:#f0f0f0;"|'''NTC:1000''' |- | BIS-DNA required (ng)||0||0 |- | JK #||0||0 |- | Conc.||0||0 |- | Volume (ul)||0.00||0.00 |- | H2O (ul)||5.50||3.99 |- | V1N3 amount (ng)||1.10||2.20 |- | V1N3 conc. (ng/ul)||0.73||0.73 |- | V1N3 volume (ul)||1.50||3.01 |- | 10X Ampligase Buffer||1.00||1.00 |- | H2O in Mix||2.00||2.00 |- | Total||10.00||10.00 |} <u>'''V1N3 & Ampligase Buffer mix</u> {| {{table}} | align="center" style="width:140px;background:#f0f0f0;"|'''Mix 0.11''' | align="center" style="width:60px;background:#f0f0f0;"|'''1x''' | align="center" style="width:60px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||0.15||0.60 |- | 10X Ampligase Buffer||1.00||4.00 |- | H2O||2.00||8.00 |- | Sum||3.15|| |} {| {{table}} | align="center" style="width:140px;background:#f0f0f0;"|'''Mix 0.22''' | align="center" style="width:60px;background:#f0f0f0;"|'''1x''' | align="center" style="width:60px;background:#f0f0f0;"|'''6x''' |- | Diluted V1N3||0.30||1.80 |- | 10X Ampligase Buffer||1.00||6.00 |- | H2O||2.00||12.00 |- | Sum||3.30|| |} {| {{table}} | align="center" style="width:140px;background:#f0f0f0;"|'''Mix 0.44''' | align="center" style="width:60px;background:#f0f0f0;"|'''1x''' | align="center" style="width:60px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||0.60||2.40 |- | 10X Ampligase Buffer||1.00||4.00 |- | H2O||2.00||8.00 |- | Sum||3.60|| |} {| {{table}} | align="center" style="width:140px;background:#f0f0f0;"|'''Mix 0.55''' | align="center" style="width:60px;background:#f0f0f0;"|'''1x''' | align="center" style="width:60px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||0.75||3.00 |- | 10X Ampligase Buffer||1.00||4.00 |- | H2O||2.00||8.00 |- | Sum||3.75|| |} {| {{table}} | align="center" style="width:140px;background:#f0f0f0;"|'''Mix 1.1''' | align="center" style="width:60px;background:#f0f0f0;"|'''1x''' | align="center" style="width:60px;background:#f0f0f0;"|'''6x''' |- | Diluted V1N3||1.50||9.00 |- | 10X Ampligase Buffer||1.00||6.00 |- | H2O||2.00||12.00 |- | Sum||4.50|| |} {| {{table}} | align="center" style="width:140px;background:#f0f0f0;"|'''Mix 2.2''' | align="center" style="width:60px;background:#f0f0f0;"|'''1x''' | align="center" style="width:60px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||3.01||12.04 |- | 10X Ampligase Buffer||1.00||4.00 |- | H2O||2.00||8.00 |- | Sum||6.01|| |} '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 1ul SLN mix (2U/ul Hemo Klentaql fragment; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 4:40h-> 94C 2min -> add 1.5ul Exo I/III mix (1ul of 20U/ul ExoI & 0.5ul of 200U/ul of ExoIII)-> 37C 2h -> 94C 2min -> 4C hold.''' == Amplification == * I used 10ul of captured DNA in total 75ul PCR reaction. :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:60px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:60px;background:#f0f0f0;"|'''20 rxn mix''' |- | Captured template||10.00||0.00 |- | 10uM AmpF6.4Sol||1.50||30.00 |- | 10uM AmpR6.3.IndX (X=57-72)||1.50||0.00 |- | 2X KAPA SYBR MM||37.50||750.00 |- | H2O||24.50||490.00 |- | Total||75.00|| |} :- Aliquot 63.5ul + 1.5ul AmpR6.3 IndX + 10ul template :- Set cycle number maximum at 27X '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x19 -> 72C 3min :{| {{table}} border = 1 | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' |- | 500:1_5ng_1||Ind_57||1000:1_5ng_1||Ind_65 |- | 500:1_5ng_2||Ind_58||1000:1_5ng_2||Ind_66 |- | 500:1_10ng_1||Ind_59||1000:1_10ng_1||Ind_67 |- | 500:1_10ng_2||Ind_60||1000:1_10ng_2||Ind_68 |- | 500:1_25ng_1||Ind_61||1000:1_25ng_1||Ind_69 |- | 500:1_25ng_2||Ind_62||1000:1_25ng_2||Ind_70 |- | 500:1_50ng_1||Ind_63||1000:1_50ng_1||Ind_71 |- | 500:1_50ng_2||Ind_64||1000:1_50ng_2||Ind_72 |} ==== PAGE verification ==== * Loaded 4ul of PCR products in 6% TBE gel [[File:ZhangLab_2 2014-06-03 16hr 20min_V1N3_500to1.jpg| 450px]] <br> <br> [[File:ZhangLab_2 2014-06-03 16hr 20min_V1N3_1000to1.jpg| 450px]] * Dr. Zhang suggested to do sequencing on 5ng and 10ng input. * I pooled PCR product by using the gel image intensity to get equimolar ratio of each libraries, including 5ng-1000:1 rep 1 &2 and 10ng-1000:1 rep1 and 2. * Purified pooled PCR products with 0.8X AMPure beads and eluted with 40ul EB buffer. * Loaded in 2-wells of 5-well 6% TBE gel [[File:ZhangLab_2 2014-06-06 09hr 53min_PAGE-SS_V1N3-5-10ng.jpg|250px]] * Resuspend sequencing libraries in 20ul H2O '''Sequencing library ID:''' NP-BSPP-MNV1N3-5-10ng, Jun1, 2014 :- Conc. by Qubit dsDNA HS = 0.644ng/ul (Alan did not have enough room for more libraries in qPCR) :- Run: HL188 (required ~300k/library) :- Length: 375bp :- Index: 65-68 (5ng-rep1, 5ng-rep2, 10ng-rep1, 10ng-rep2) :- They all used probe:target = 1000:1 :{| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' |- | Ind_65||JK_5ng_1 |- | Ind_66||JK_5ng_2 |- | Ind_67||JK_10ng_1 |- | Ind_68||JK_10ng_2 |} :- JK = Jurkat DNA
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