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= MONOD V1N3 BSPP capture = '''Link to calendar: [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014]]''' * I will do BSPP capture using the new probe set, MONOD V1N3. This probe set, Dr. Zhang selected the probes with high capture efficiency from GP1V4 and MONOD V3. The size of probe is quite small ~2400 probes. * I am going to test capture sensitivity on different amount of BIS-JURKAT DNA input at 5, 10, 25, and 50ng with 200ng as positive control * To test the recovery rate after bisulfite conversion, I perform bisulfite conversion at different amount of gDNA. == Bisulfite conversion == * Received new tube of JURKAT DNA. * Re-quantified concentration with Qubit dsDNA assay HS (1ul in 200ul reaction = 200X dilution) ** Conc. = 82.1ng/ul * Amount/ # of tube for bisulfite conversion ** 100ng/ 3tubes ** 200ng/ 3tubes ** 600ng/ 2tubes :{| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:90px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:90px;background:#f0f0f0;"|'''Amount (ng)''' | align="center" style="width:90px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:90px;background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="width:90px;background:#f0f0f0;"|'''Lightnig Conversion reagent''' | align="center" style="width:90px;background:#f0f0f0;"|'''Total''' |- | JK-1||82.10||100.00||1.22||18.78||130.00||150.00 |- | JK-2||82.10||100.00||1.22||18.78||130.00||150.00 |- | JK-3||82.10||100.00||1.22||18.78||130.00||150.00 |- | JK-4||82.10||200.00||2.44||17.56||130.00||150.00 |- | JK-5||82.10||200.00||2.44||17.56||130.00||150.00 |- | JK-6||82.10||200.00||2.44||17.56||130.00||150.00 |- | JK-7||82.10||600.00||7.31||12.69||130.00||150.00 |- | JK-8||82.10||600.00||7.31||12.69||130.00||150.00 |} * Follow the instruction of the kit * Elute with 17ul Elution buffer * Volume left is ~15ul * Quantified concentration of BIS- JURKAT DNA with Qubit ssDNa assay (1ul in 200ul reaction = 200x dilution) ==== ssDNA Qubit assay result ==== :{| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:90px;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:90px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:90px;background:#f0f0f0;"|'''Sample conc. (ng/ul)''' | align="center" style="width:90px;background:#f0f0f0;"|'''Yield in 15ul (ng)''' | align="center" style="width:90px;background:#f0f0f0;"|'''Input DNA (ng)''' | align="center" style="width:90px;background:#f0f0f0;"|'''% Recovery''' |- | JK-1||15.2||ng/mL||1||200||3.03||45.45||100||45.45 |- | JK-2||19.1||ng/mL||1||200||3.82||57.30||100||57.30 |- | JK-3||13.3||ng/mL||1||200||2.66||39.90||100||39.90 |- | JK-4||41||ng/mL||1||200||8.21||123.15||200||61.58 |- | JK-5||36.6||ng/mL||1||200||7.31||109.65||200||54.83 |- | JK-6||39.4||ng/mL||1||200||7.88||118.20||200||59.10 |- | JK-7||133||ng/mL||1||200||26.6||399.00||600||66.50 |- | JK-8||131||ng/mL||1||200||26.2||393.00||600||65.50 |} :{| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''DNA input''' | align="center" style="width:100px;background:#f0f0f0;"|'''Ave. % recovery rate''' | align="center" style="width:100px;background:#f0f0f0;"|'''STDEV''' |- | 100ng||47.55||8.89 |- | 200ng||58.5||3.41 |- | 600ng||66||0.71 |} * It seems like recovery rate is proportional to DNA input. However, I could expect at least 40% from 100ng input. I should try with lower amount of DNA (25 or 50ng) since cell-free DNA from cancer samples have very low DNA amount and varied between different samples. == BSPP capture set up == * BIS-DNA: 5ng, 10ng, 25ng, 50ng and 200ng as PC * Probe-to-target: 1,000:1 & 2,000:1 ==== Probe capculation ==== :{| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Template (ng)''' | align="center" style="width:120px;background:#f0f0f0;"|'''ratio''' | align="center" style="width:120px;background:#f0f0f0;"|'''V1N3 amount required (ng)''' |- | 200||1,000:1||8.80 |- | 200||2,000:1||17.61 |- | |||| |- | 5||1,000:1||0.22 |- | 5||2,000:1||0.44 |- | |||| |- | 10||1,000:1||0.44 |- | 10||2,000:1||0.88 |- | |||| |- | 25||1,000:1||1.10 |- | 25||2,000:1||2.20 |- | |||| |- | 50||1,000:1||2.20 |- | 50||2,000:1||4.40 |} ==== V1N3 capture set up ==== * '''X-XXXX''' = '''BIS-DNA input amount-probe-to-target ratio''' {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''strip 1''' | align="center" style="width:90px;background:#f0f0f0;"|'''5-1000''' | align="center" style="width:90px;background:#f0f0f0;"|'''5-1000''' | align="center" style="width:90px;background:#f0f0f0;"|'''10-1000''' | align="center" style="width:90px;background:#f0f0f0;"|'''10-1000''' | align="center" style="width:90px;background:#f0f0f0;"|'''25-1000''' | align="center" style="width:90px;background:#f0f0f0;"|'''25-1000''' | align="center" style="width:90px;background:#f0f0f0;"|'''50-1000''' | align="center" style="width:90px;background:#f0f0f0;"|'''50-1000''' |- | BIS-DNA required (ng)||5||5||10||10||25||25||50||50 |- | JK #||JK-1||JK-1||JK-2||JK-2||JK-4||JK-4||JK-5||JK-5 |- | Conc.||3.03||3.03||3.82||3.82||8.21||8.21||7.31||7.31 |- | Volume (ul)||1.65||1.65||2.62||2.62||3.05||3.05||6.84||6.84 |- | H2O (ul)||13.05||13.05||11.78||11.78||10.45||10.45||5.15||5.15 |- | 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||2.00||2.00||2.00||2.00||2.00||2.00||2.00||2.00 |- | H2O in Mix||3.00||3.00||3.00||3.00||3.00||3.00||3.00||3.00 |- | Total||20.00||20.00||20.00||20.00||20.00||20.00||20.00||20.00 |} {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''strip 2''' | align="center" style="width:90px;background:#f0f0f0;"|'''5-2000''' | align="center" style="width:90px;background:#f0f0f0;"|'''5-2000''' | align="center" style="width:90px;background:#f0f0f0;"|'''10-2000''' | align="center" style="width:90px;background:#f0f0f0;"|'''10-2000''' | align="center" style="width:90px;background:#f0f0f0;"|'''25-2000''' | align="center" style="width:90px;background:#f0f0f0;"|'''25-2000''' | align="center" style="width:90px;background:#f0f0f0;"|'''50-2000''' | align="center" style="width:90px;background:#f0f0f0;"|'''50-2000''' |- | BIS-DNA required (ng)||5||5||10||10||25||25||50||50 |- | JK #||JK-1||JK-1||JK-2||JK-2||JK-4||JK-4||JK-6||JK-6 |- | Conc.||3.03||3.03||3.82||3.82||8.21||8.21||7.88||7.88 |- | Volume (ul)||1.65||1.65||2.62||2.62||3.05||3.05||6.35||6.35 |- | H2O (ul)||12.75||12.75||11.18||11.18||8.95||8.95||2.64||2.64 |- | V1N3 amount (ng)||0.44||0.44||0.88||0.88||2.20||2.20||4.40||4.40 |- | V1N3 conc. (ng/ul)||0.73||0.73||0.73||0.73||0.73||0.73||0.73||0.73 |- | V1N3 volume (ul)||0.60||0.60||1.20||1.20||3.01||3.01||6.02||6.02 |- | 10X Ampligase Buffer||2.00||2.00||2.00||2.00||2.00||2.00||2.00||2.00 |- | H2O in Mix||3.00||3.00||3.00||3.00||3.00||3.00||3.00||3.00 |- | Total||20.00||20.00||20.00||20.00||20.00||20.00||20.00||20.00 |} {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Strip 3''' | align="center" style="width:90px;background:#f0f0f0;"|'''200-1000''' | align="center" style="width:90px;background:#f0f0f0;"|'''200-2000''' | align="center" style="width:90px;background:#f0f0f0;"|'''NTC-200-1000''' | align="center" style="width:90px;background:#f0f0f0;"|'''NTC-200-2000''' |- | BIS-DNA required (ng)||200||200||0||0 |- | JK #||JK-7||JK-8||NA||NA |- | Conc.||26.60||26.20||0.00||0.00 |- | Volume (ul)||7.52||7.63||0.00||0.00 |- | H2O (ul)||9.18||7.76||16.70||15.39 |- | V1N3 amount (ng)||8.80||17.61||8.80||17.61 |- | V1N3 conc. (ng/ul)||6.75||6.75||6.75||6.75 |- | V1N3 volume (ul)||1.30||2.61||1.30||2.61 |- | 10X Ampligase Buffer||2.00||2.00||2.00||2.00 |- | Total||20.00||20.00||20.00||20.00 |} ==== Probe & buffer reaction mix ==== * I diluted 10x of V1N3 by mixing 8ul of V1N3 with 72ul H2O and re-quantified concentration with Qubit ssDNA assay: <u>0.73ng/ul</u> *'''Mix XX = amount of V1N3 in each reaction''' :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 0.22''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||0.30||1.20 |- | 10X Ampligase Buffer||2.00||8.00 |- | H2O||3.00||12.00 |- | Sum||5.30|| |} :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 0.44''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''6x''' |- | Diluted V1N3||0.60||3.60 |- | 10X Ampligase Buffer||2.00||12.00 |- | H2O||3.00||18.00 |- | Sum||5.60|| |} :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 0.88''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||1.20||4.80 |- | 10X Ampligase Buffer||2.00||8.00 |- | H2O||3.00||12.00 |- | Sum||6.20|| |} :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 1.1''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||1.50||6.00 |- | 10X Ampligase Buffer||2.00||8.00 |- | H2O||3.00||12.00 |- | Sum||6.50|| |} :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 2.2''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''6X''' |- | Diluted V1N3||3.01||18.06 |- | 10X Ampligase Buffer||2.00||12.00 |- | H2O||3.00||18.00 |- | Sum||8.01|| |} :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 4.4''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||6.02||24.08 |- | 10X Ampligase Buffer||2.00||8.00 |- | H2O||3.00||12.00 |- | Sum||11.02|| |} '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16h -> add 2ul SLN mix (2U/ul Hemo KlenTaq; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 19h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 5min -> 4C hold. ==== SLN Mix ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Stock conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Prepare volume, total 48ul''' |- | Hemo KlenTaq||10||U/ul||2||U/ul||9.60 |- | AmpLigase||5||U/ul||0.5||U/ul||4.80 |- | dNTP||1||mM||100||uM||4.80 |- | 10x AmpLigase Buffer||10||x||1||x||4.80 |- | H2O||||||||||24.00 |} * Aliquot 12ul to 4-tube strip == Amplification == * I did PCR using 20ul of captured DNA in total 100ul reaction. For 200ng input and it's NTC, I used only 10ul because I know that it will shoot up earlier than other reaction. {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:100px;background:#f0f0f0;"|'''21.5 rxn mix''' |- | Captured template||20.00||0.00 |- | 10uM AmpF6.4Sol||2.00||43.00 |- | 10uM AmpR6.3.IndX (X=73-93)||2.00||0.00 |- | 2X KAPA SYBR MM||50.00||1,075.00 |- | H2O||26.00||559.00 |- | Total||100.00|| |} * Aliquot 78ul + 2ul AmpR6.3 IndX + 20ul template '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x19 -> 72C 3min *I stopped each reaction at different cycles number based on qPCR curves to get optimum intensity of expected band ** <u>Strip 1:</u> *** 5-1000, 10-1000: 24X *** 25-1000, 50-1000: 22X ** <u>Strip 1:</u> *** 5-2000, 10-2000: 24X *** 25-2000: 21X ***50-2000: 22X ** <u>Strip 3:</u> *** 200-100, 200-2000, NTC-2000, NTC PCR : 20X *** NTC-1000: 24X ==== PAGE verification ==== * Loaded 4ul of PCR product in 6% TBE gel '''1000:1 probe:target'''<br> [[File:ZhangLab_2 2014-05-31 18hr 23min_MONOD.V1N3_gel1_1-1000.jpg| 500px]]<br> <br> '''2000:1 probe:target'''<br> [[File:ZhangLab_2 2014-05-31 18hr 23min_MONOD.V1N3_gel2_1to2000.jpg| 500px]] * From the gel images above, I can observe a clear smear in at least 50ng of DNA BIS-DNA either 1000:1 and 2000:1 probe to target ratio. * 200ng input showed not too strong intensity because I used only 10% v/v in PCR and stopped earlier than other samples. * For lower amount, it's not very clear that the smear ~30bp or above is the captured target as it did not show s sharp smear border as observed in 50ng and 200ng. * I still wanted to test more on 10 and 25ng input since some of DNA extracted from serum of cancer patient is lower than 50ng. After bisulfite conversion, the amount of DNa should be even lower than 25ng. * I may repeat capture in smaller volume 10ul by including 10m 25 and 50ng. * I wanted to reduced incubation time after adding SLN mix to 6-12h as too long incubation with polymerase may be unnecessary and causing some background for the low input DNA. * I could not explain why I alway get a weird pattern like in gel1 lane 4 and gel2 lane 4.
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