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==Procedures== *Prepare ice box. Clean the PCR hood. Set up realtime PCR program. *UV the following tubes: #PCR tube strip x1(Bio-rad) cap x1(Bio-rad) #0.5mL tube x1 (for master mix) #Cold block *Centrifuge the ligation products. 2min, 10000rpm, RT. Leave on ice. *Thaw the primers:N2-indexed-Fs(100uM) and N2-indexed-10R(100uM). Diluted them into 10uM (5uL stock sol. + 45uL H2O) *Thaw 2X Phusion HF enzyme mix *Setup the qPCR program. *Lib-amp PCR(100uL): (modified from Dr.Zhang recipe) 1rxn 2.5 rxn H2O 34 85.0 uL Adapter ligated DNA 10 - uL PCR_F(10uM) 2 5.0 uL PCR_R.N2 Index(10uM) 2 - uL --- using N2-indexed-primer "10" 20X EVA Green 2 5.0 uL 2X Phusion HF MM 50 125.0 uL ------------------ 100 220.0/2.5=88 --- template 10uL, Index primer(R) 2uL *NOTE1: I also amplify another library c6 to balance with c5. *NOTE2: '''N2-index-(10)R''' primer was used for both C5 and C6 libraries. *The reactions tubes were pre-cut before incubation. **Using new two-step PCR program. qPCR was performed using Eppendorf realplex machine: 98C 30sec -> (98C 10sec -> 65C 20sec -> 72C 20sec) x 8 cycles -> (98C 10sec -> 72C 20sec) x 10 cycles -> 72C 3min -> hold 15C. *I will expect the stop the reactions around 15th~16th cycle or the monitored dif. of Flouresscensce reach 14000. Depends on which one reach first. *Aliquote 3uL of amplified library for TBE-PAGE validation later. *Use AMpure beads(1V, 100uL) to purify the rest of libraries. Elute in 20uL H2O. Aliquote 1uL of purified library for TBE-PAGE validation. **In AMpure bead manual, 1.8V beads are recommended. *For TBE-PAGE validation. Different sample amount were loaded since purified sample is much more concentrated, and I don't want to waste it on gel loading. **Un-purified samples(3uL) and purified sample(1uL). *TBE-PAGE **Unpurified sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE buffer **Puriried samplewell : 1uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer **Ladder well : 1uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer **Gel was run at 200V for 30min. **Use 3uL SYBR-gold with regular stainig(5~10min) with foil protection
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