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== Amplification == === Things to consider === * To prepare at least 500ng up to 1000ng amount WGBS library from low input DNA, I alway do two-rounds of amplification. This is different from preparing WGBS libraries for sequencing which requires less than 100ng of each library for sequencing. * From my previous experiments, running qPCR in small volume (50ul) using all DNA from bisulfite conversion (input 1-10ng) was saturated with the yield less than 100ng). To be able to get almost up to 1ug of WGBS library, I need to set up the PCR volume up to 300ul (decreasing DNA template concentration) and amplify with more cycle number (12-18 cycles for input between 1ng-20ng, varied based on input DNA). I have considered that using 2X KAPA HiFi HotStart Uracil+ ReadyMix for qPCR reaction 300ul is very expensive for a routine experiment and also to set up a protocol for large sample size. I then have tried to do two rounds of qPCR by using 2X KAPA HiFi HotStart Uracil+ ReadyMix in the first round qPCR in total reaction 50ul. I then do 1X bead purification and amplify the 2nd round PCR using other high efficient DNA Pol with proof reading activity like NEB Phusion HiFidelity or KAPA HiFi Hotstart Readymix. However, this two-step PCR is not convenient as well and AMPure bead is quite more expensive. I may need to judge between the cost and time between using one round and two-round PCR. * For Illumina, the cost is not a limitation, so I suggested Li to do one round qPCR by increasing amplification volume and increase cycle number. This should save a lot of time. ==== Quick test PCR to monitor PCR cycle number ==== * I use 1ul of bisulfite converted DNA in 10ul reaction for a quick test and will use this condition for large volume amplification after obtaining optimal PCR cycle number {| {{table}} border = 1 | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1X 50ul rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''10ul rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''12X 10ul rxn''' |- | Adaptor ligated DNA||5.00||1.00||0.00 |- | 2X KAPA HiFi HotStart Uracil+ ReadyMix||25.00||5.00||60.00 |- | 10uM TruS F/R||1.50||0.30||3.60 |- | 50X SYBR||0.40||0.08||0.96 |- | H2O||18.10||3.62||43.44 |- | Total ||50.00||10.00|| |} :- Aliquot 9ul + 1ul template :- Mix well ::''98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 8-18X --> 72C for 1min'' :- Load 3ul PCR product in 6% TBE gel to verify the amplified libraries [[File:ZhangLab_2 2014-12-05 16hr 57min_ILMN_qPCR-quicktest.jpg| 450px]] * Note: qPCR curves of KU_2 and KU_6 shoot up later than other samples with the same input amount. KU_2 curve was very close to NTC, but it had good amplification at 18 cycles. The color (orange) appeared in this sample may interfere fluorescence reading. ==== Amplification in a large volume ==== * After quick test qPCR and PAGE verification, I continued to amplify the rest of bisulfite converted DNA in total 300ul (split 50ul/tube) by adjusting PCR cycle number for some samples regarding gel intensity. {| {{table}} border = 1 | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1X 50ul rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''1X 150''' | align="center" style="width:80px;background:#f0f0f0;"|'''21 rxn''' |- | Adaptor ligated DNA||5.00||15.00||0.00 |- | 2X KAPA HiFi HotStart Uracil+ ReadyMix||25.00||75.00||1575.00 |- | 10uM TruS F/R||1.50||4.50||94.50 |- | 50X SYBR||0.40||1.20||25.20 |- | H2O||18.10||54.30||1140.30 |- | Total ||50.00||150.00|| |} :- Aliquot 135ul + 15ul template (2X of 150ul -> then split 50ul/tube) :- Mix well ::''98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 12-18X --> 72C for 1min'' <br> {| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Input ampiunt''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of cycle''' |- | KU_1||4.6||15 |- | KU_2||4.8||18 |- | KU_3||5.1||15 |- | KU_4||8.6||15 |- | KU_5||9||14 |- | KU_6||9.57||16 |- | KU_7||11.1||14 |- | KU_8||11.9||12 |- | KU_9||13.2||12 |- | KU_10||14||12 |} :- Perform 1X of AMPure bead purification by pooling 100ul of PCR product of the same sample in each tube (3 tubes/sample) :- Elute each tube with 25ul H2O -> total volume of each sample is ~75ul. :- I aliquot 2ul for Li for BA measurement. :- I took 1ul for Qubit dsDNA HS assay. :- The total volume left for each sample should be ~70ul ==== Qubit quantification assay results ==== {| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;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:80px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 70ul (ng)''' |- | KU_1||49.4||ng/mL||1||200||9.89||692.30 |- | KU_2||74.4||ng/mL||1||200||14.90||1043.00 |- | KU_3||54.8||ng/mL||1||200||11.00||770.00 |- | KU_4||71.5||ng/mL||1||200||14.30||1001.00 |- | KU_5||50.6||ng/mL||1||200||10.10||707.00 |- | KU_6||63.5||ng/mL||1||200||12.70||889.00 |- | KU_7||78.2||ng/mL||1||200||15.60||1092.00 |- | KU_8||46.1||ng/mL||1||200||9.22||645.40 |- | KU_9||45.5||ng/mL||1||200||9.09||636.30 |- | KU_10||40.8||ng/mL||1||200||8.17||571.90 |} ==== Image of Bioanalyzer By Li ==== [[File:Beadpurified-WGBSLib-BA.png|800px]]
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