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= BSPP capture of amplified DNA from Illumina using 3 different enzymes for polymerization = [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014 Link to calendar]]<br> * From previous experiment on 2014-05-13 [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-5-13]], the results were inconclusive, and I lacked positive control. Dr. Zhang suggested to repeat experiment on few samples from Illumina and include cancer sample DNA as control. * I need to prepare more GP1V4 BSPP. ==== Sample list and experimental design ==== * Cancer sample #1 or C1 (BXPC3) * Cancer sample #2 or C2 (U87MG) * Illumina amplified DNA, A1 * Illumina amplifed DNA, A2 * Capture with 500:1 GP1V4:target ratio (total amount in each reaction ~13ng) * Bisulfite treated DNA/amplified DNA amount 200ng * Add different enzyme in SLN mix, ** Stoffel (blue color) ** Hemo KlenTaq (red color) ** Illumina Polymerase (black color) * I include sample C1, A1, and A2 in capture reaction, but not include GP1V4. This is may not necessary, but I want to make sure that there is no any background from the template itself. * For cancer samples, I performed bisulfite conversion 6tubes of C1 and 3tubes of C2 samples (600ng gDNA for each tube). I used bisulfite treated DNA from different tubes for each reaction. There might me some variations of DNA amount in each reaction based on accuracy of DNA concentration quantification or pipetting. == Bisulfite conversion == * I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo and followed the protocol of the kit * Follow the instruction of the kit '''Program''' 1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold * Elute with 16ul elution buffer. Total volume left after elution ~15ul * Took 2ul for Qubit ssDNA assay :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Concentration in the Qubit''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''uL used''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''Sample Concentration''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Yield in 15ul (ng)''' | align="center" style="background:#f0f0f0;"|'''% Recovery''' |- | CANC1-1||219||ng/mL||2||100||21.9||ug/mL||328.50||54.75 |- | CANC1-2||204||ng/mL||2||100||20.4||ug/mL||306.00||51.00 |- | CANC1-3||231||ng/mL||2||100||23.1||ug/mL||346.50||57.75 |- | CANC1-4||209||ng/mL||2||100||20.9||ug/mL||313.50||52.25 |- | CANC1-5||226||ng/mL||2||100||22.6||ug/mL||339.00||56.50 |- | CANC1-6||227||ng/mL||2||100||22.7||ug/mL||340.50||56.75 |- | CANC2-1||207||ng/mL||2||100||20.7||ug/mL||310.50||51.75 |- | CANC2-2||195||ng/mL||2||200||19.5||ug/mL||292.50||48.75 |- | CANC2-3||209||ng/mL||2||100||20.9||ug/mL||313.50||52.25 |} == BSPP capture set up == * BSC_A1 conc. = 120ng/ul * BSC_A2 conc. = 100ng/ul * GP1V4 conc. (prep 2014-05-20) = 8.37ng/ul ==== Probe & AmpLigase Buffer mix ==== ===== Mix I. Amligase buffer and GP1V4 ===== :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''18 rxn mix''' |- | GP1V4 (8.37ng/ul)||1.55||27.96 |- | 10X AmpLig Buffer||2.00||36.00 |- | H2O||5.00||90.00 |- | Total||8.55|| |} ===== Mix II. only Amligase buffer ===== :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''11 rxn mix''' |- | 10X AmpLig Buffer||2.00||22.00 |- | H2O||5.00||55.00 |- | Total||7.00|| |} {| {{table}} class = wikitable | align="center" style="background:blue;"|'''Stoffel''' | align="center" style="width:50px; background:#f0f0f0;"|'''C1-S''' | align="center" style="width:50px; background:#f0f0f0;"|'''C2-S''' | align="center" style="width:50px; background:#f0f0f0;"|'''A1-S''' | align="center" style="width:50px; background:#f0f0f0;"|'''A2-S''' | align="center" style="width:50px; background:#f0f0f0;"|'''NTC''' | align="center" style="width:50px; background:#f0f0f0;"|'''C1 no GP1V4''' | align="center" style="width:50px; background:#f0f0f0;"|'''A1 no GP1V4''' | align="center" style="width:50px; background:#f0f0f0;"|'''A2 no GP1V4''' |- | Conc. (ng/ul)||21.90||20.70||120.00||100.00||||20.40||120.00||100.00 |- | Vol. for 200ng||9.13||9.66||1.67||2.00||0.00||9.80||1.67||2.00 |- | GP1V4 (8.37ng/ul)||1.55||1.55||1.55||1.55||1.55||0.00||0.00||0.00 |- | H2O||2.31||1.78||9.78||9.45||11.45||3.20||11.33||11.00 |- | 10X Ampligase buffer||2.00||2.00||2.00||2.00||2.00||2.00||2.00||2.00 |- | H2O in Mix||5.00||5.00||5.00||5.00||5.00||5.00||5.00||5.00 |- | Total||20.00||20.00||20.00||20.00||20.00||20.00||20.00||20.00 |} {| {{table}} class = wikitable | align="center" style="background:red;"|'''Hemo Klentaq''' | align="center" style="width:50px; background:#f0f0f0;"|'''C1-S''' | align="center" style="width:50px; background:#f0f0f0;"|'''C2-S''' | align="center" style="width:50px; background:#f0f0f0;"|'''A1-S''' | align="center" style="width:50px; background:#f0f0f0;"|'''A2-S''' | align="center" style="width:50px; background:#f0f0f0;"|'''NTC''' | align="center" style="width:50px; background:#f0f0f0;"|'''C1 no GP1V4''' | align="center" style="width:50px; background:#f0f0f0;"|'''A1 no GP1V4''' | align="center" style="width:50px; background:#f0f0f0;"|'''A2 no GP1V4''' |- | Conc. (ng/ul)||23.10||19.50||120.00||100.00||||20.90||120.00||100.00 |- | Vol. for 200ng||8.66||10.26||1.67||2.00||0.00||9.57||1.67||2.00 |- | GP1V4 (8.37ng/ul)||1.55||1.55||1.55||1.55||1.55||0.00||0.00||0.00 |- | H2O||2.79||1.19||9.78||9.45||11.45||3.43||11.33||11.00 |- | 10X Ampligase buffer||2.00||2.00||2.00||2.00||2.00||2.00||2.00||2.00 |- | H2O in Mix||5.00||5.00||5.00||5.00||5.00||5.00||5.00||5.00 |- | Total||20.00||20.00||20.00||20.00||20.00||20.00||20.00||20.00 |} {| {{table}} class =wikitable | align="center" style="background:dimgray;"|'''ILMN-Pol''' | align="center" style=width:50px; "background:#f0f0f0;"|'''C1-S''' | align="center" style=width:50px; "background:#f0f0f0;"|'''C2-S''' | align="center" style=width:50px; "background:#f0f0f0;"|'''A1-S''' | align="center" style=width:50px; "background:#f0f0f0;"|'''A2-S''' | align="center" style=width:50px; "background:#f0f0f0;"|'''NTC''' | align="center" style=width:50px; "background:#f0f0f0;"|'''C1 no GP1V4''' | align="center" style=width:50px; "background:#f0f0f0;"|'''A1 no GP1V4''' | align="center" style=width:50px; "background:#f0f0f0;"|'''A2 no GP1V4''' |- | Conc. (ng/ul)||22.60||20.90||120.00||100.00||||22.70||120.00||100.00 |- | Vol. for 200ng||8.85||9.57||1.67||2.00||0.00||8.81||1.67||2.00 |- | GP1V4 (8.37ng/ul)||1.55||1.55||1.55||1.55||1.55||0.00||0.00||0.00 |- | H2O||2.60||1.88||9.78||9.45||11.45||4.19||11.33||11.00 |- | 10X Ampligase buffer||2.00||2.00||2.00||2.00||2.00||2.00||2.00||2.00 |- | H2O in Mix||5.00||5.00||5.00||5.00||5.00||5.00||5.00||5.00 |- | Total||20.00||20.00||20.00||20.00||20.00||20.00||20.00||20.00 |} '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2ul SLN mix (2U/ul '''Polymerase''' (NEB); 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. * For Illumina's polymerase I used 1ul for each sample as recommended by Xuyu ==== SLN mix solution ==== {| {{table}} | 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 (ul), total 20ul''' |- | Stoffel/ Hemo KlenTaq||10||U/ul||2||U/ul||4.00 |- | AmpLigase||5||U/ul||0.5||U/ul||2.00 |- | dNTP||1||mM||100||uM||2.00 |- | 10x AmpLigase Buffer||10||x||1||x||2.00 |- | H2O||||||||||10.00 |} {| {{table}} | 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 (ul), total 20ul''' |- | Illumina's Polymerase||3||U/ul||1.5||U/ul||10.00 |- | AmpLigase||5||U/ul||0.5||U/ul||2.00 |- | dNTP||1||mM||100||uM||2.00 |- | 10x AmpLigase Buffer||10||x||1||x||2.00 |- | H2O||||||||||4.00 |} == PCR Amplification == * I performed PCR on half of captured DNA (10ul) in total 50ul PCR reaction (20% v/v in PCR) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''26 rxn mix''' |- | Captured template||10.00||0.00 |- | 10uM AmpF6.4Sol||1.00||26.00 |- | 10uM AmpR6.3.IndX (X=1-24)||1.00||0.00 |- | 2X KAPA SYBR MM||25.00||650.00 |- | H2O||13.00||338.00 |- | Total||50.00|| |} * Aliquot 39ul + 1ul AmpR6.3 IndX + 10ul DNA template {| {{table}} class =wikitable | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="width:50px; background:#f0f0f0;"|'''C1''' | align="center" style="width:50px; background:#f0f0f0;"|'''C2''' | align="center" style="width:50px; background:#f0f0f0;"|'''A1''' | align="center" style="width:50px; background:#f0f0f0;"|'''A2''' | align="center" style="width:50px; background:#f0f0f0;"|'''NTC''' | align="center" style="width:50px; background:#f0f0f0;"|'''C1 no GP1V4''' | align="center" style="width:50px; background:#f0f0f0;"|'''A1 no GP1V4''' | align="center" style="width:50px; background:#f0f0f0;"|'''A2 no GP1V4''' |- | Strip 1: Stoffel||Ind_1||Ind_2||Ind_3||Ind_4||Ind_5||Ind_6||Ind_7||Ind_8 |- | Strip 2: Hemo KlenTaq||Ind_9||Ind_10||Ind_11||Ind_12||Ind_13||Ind_14||Ind_15||Ind_16 |- | Strip 3: ILMN's Pol||Ind_17||Ind_18||Ind_19||Ind_20||Ind_21||Ind_22||Ind_23||Ind_24 |} '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min * I stopped all samples at 21x cycles except sample C1,C2 with Hemo KlenTaq/ Illumina' Polymerase at 22x cycles. * Verify 3ul of PCR product in 6% TBE gel (no AMPure bead purified) '''Stoffel Fragment'''<br> [[File:ZhangLab_2 2014-05-26 13hr 01min_GP1v4_C1C2A1A2NTC_Stoffel.jpg| 400px]]<br> '''Hemo KlenTaq'''<br> [[File:ZhangLab_2 2014-05-26 13hr 01min_GP1v4_C1C2A1A2ntc_HemoKlenTaq.jpg| 400px]]<br> '''Illumina's Polymerase'''<br> [[File:ZhangLab_2 2014-05-26 13hr 01min_GP1v4_C1C2A1A2ntc_ILPol.jpg| 400px]]<br> * ** = over-amplified * From the result above, it's clear that cancer samples with '''Stoffel fragment''' and '''Hemo Klentaq''' showed a smear between 350~390bp. I think there is the variation of DNA input in different tubes because there is more background in the samples with strong signal. * Amplified DNA from Illumina showed the same pattern in three enzyme, but has highest background in Hemo KlenTaq * With Illumina's polymerase, cancer samples showed discrete bands of ~350, 390 and above 400bp similar to amplified DNA from Illumina. ---- * Example of GP1V4 capture on cancer and blood samples I did experiment on 2013-12-24 ('''after AMPure bead purified''') [[File:ZhangLab_2 2013-12-25 19hr 44min_GP1_V4_1-8_PQ.jpg| 400px]]
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