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==== 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
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