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=== Plate layout and barcoded primer === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|A1 | align="center" style="background:#f0f0f0;"|A2 | align="center" style="background:#f0f0f0;"|A3 | align="center" style="background:#f0f0f0;"|A4 | align="center" style="background:#f0f0f0;"|A5 | align="center" style="background:#f0f0f0;"|A6 | align="center" style="background:#f0f0f0;"|A7 | align="center" style="background:#f0f0f0;"|A8 |- | AmpR6.3.Ind1||AmpR6.3.Ind9||AmpR6.3.Ind17||AmpR6.3.Ind25||AmpR6.3.Ind33||AmpR6.3.Ind41||AmpR6.3.Ind49||AmpR6.3.Ind57 |- | B1||B2||B3||B4||B5||B6||B7||B8 |- | AmpR6.3.Ind2||AmpR6.3.Ind10||AmpR6.3.Ind18||AmpR6.3.Ind26||AmpR6.3.Ind34||AmpR6.3.Ind42||AmpR6.3.Ind50||AmpR6.3.Ind58 |- | C1||C2||C3||C4||C5||C6||C7||C8 |- | AmpR6.3.Ind3||AmpR6.3.Ind11||AmpR6.3.Ind19||AmpR6.3.Ind27||AmpR6.3.Ind35||AmpR6.3.Ind43||AmpR6.3.Ind51||AmpR6.3.Ind59 |- | D1||D2||D3||D4||D5||D6||D7||D8 |- | AmpR6.3.Ind4||AmpR6.3.Ind12||AmpR6.3.Ind20||AmpR6.3.Ind28||AmpR6.3.Ind36||AmpR6.3.Ind44||AmpR6.3.Ind52||AmpR6.3.Ind60 |- | E1||E2||E3||E4||E5||E6||E7||E8 |- | AmpR6.3.Ind5||AmpR6.3.Ind13||AmpR6.3.Ind21||AmpR6.3.Ind29||AmpR6.3.Ind37||AmpR6.3.Ind45||AmpR6.3.Ind53||AmpR6.3.Ind61 |- | F1||F2||F3||F4||F5||F6||F7||F8 |- | AmpR6.3.Ind6||AmpR6.3.Ind14||AmpR6.3.Ind22||AmpR6.3.Ind30||AmpR6.3.Ind38||AmpR6.3.Ind46||AmpR6.3.Ind54||AmpR6.3.Ind62 |- | G1||G2||G3||G4||G5||G6||G7|| |- | AmpR6.3.Ind7||AmpR6.3.Ind15||AmpR6.3.Ind23||AmpR6.3.Ind31||AmpR6.3.Ind39||AmpR6.3.Ind47||AmpR6.3.Ind55|| |- | H1||H2||H3||H4||H5||H6||H7|| |- | AmpR6.3.Ind8||AmpR6.3.Ind16||AmpR6.3.Ind24||AmpR6.3.Ind32||AmpR6.3.Ind40||AmpR6.3.Ind48||AmpR6.3.Ind56|| |} * Note: There are total 70 reactions (A1-H9, no samples in well ID G8 and H8 and A10-H12) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''71 rxn mix''' |- | Captured template||10.00||0.00 |- | 10uM AmpF6.4Sol||2.00||142.00 |- | 10uM AmpR6.3Indx (X=1-72)||2.00||0.00 |- | 2x Kapa SYBR MM||50.00||3550.00 |- | H2O||36.00||2,556.00 |- | Total||100.00||6,248.00 |} * Aliquot 88ul, add 2ul of 10uM AmpR6.3Indx and 10ul of circularized DNA template '''Program'''<br> 98C 30s -> (98C 10s -> 58C 30s -> 72C 30s)x8 -> (98C 10s -> 72C 30s)x14 -> 72C 3min * I performed qPCR in duplicate with the same condition and stopped at the same cycle [[File:2012_05_25_qPCR_SR_plate1.png| 500px]] [[File:2012_05_25_qPCR_SR_plate2.png| 500px]] * Since A9-H9 reactions shoot up earlier than other samples, I took out 60ul at cycles 20 - Took 60ul of PCR product from plate1 and 2 (total volume 120ul) - Purified with 0.8 vol of AmPure beads - Eluted with 60ul EB buffer - Performed PAGE quantification by loading sample 1ul {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Well ID''' | align="center" style="background:#f0f0f0;"|'''conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount in 60ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 60ng (ul)''' | align="center" style="background:#f0f0f0;"|'''Well ID''' |- | A1||13.66||819.34||4.39||A1 |- | B1||11.97||718.02||5.01||B1 |- | C1||12.50||750.04||4.80||C1 |- | D1||11.37||682.37||5.28||D1 |- | E1||9.32||559.04||6.44||E1 |- | F1||11.67||699.95||5.14||F1 |- | G1||12.32||739.22||4.87||G1 |- | H1||13.97||838.08||4.30||H1 |- | A2||14.19||851.55||4.23||A2 |- | B2||13.07||784.08||4.59||B2 |- | C2||14.71||882.63||4.08||C2 |- | D2||14.17||850.27||4.23||D2 |- | E2||17.12||1026.97||3.51||E2 |- | F2||16.72||1003.03||3.59||F2 |- | G2||17.42||1045.27||3.44||G2 |- | H2*||15.41||924.62||3.89||H2* |- | A3||14.31||858.62||4.19||A3 |- | B3||16.01||960.41||3.75||B3 |- | C3||15.83||949.64||3.79||C3 |- | D3||10.69||641.17||5.61||D3 |- | E3||6.92||415.03||8.67||E3 |- | F3||12.93||775.83||4.64||F3 |- | G3||12.04||722.32||4.98||G3 |- | H3||12.10||726.03||4.96||H3 |- | A4||16.39||983.11||3.66||A4 |- | B4||15.02||901.02||4.00||B4 |- | C4||12.66||759.53||4.74||C4 |- | D4||9.03||541.89||6.64||D4 |- | E4||16.28||976.88||3.69||E4 |- | F4||11.63||698.04||5.16||F4 |- | G4*||8.79||527.68||6.82||G4* |- | H4||17.56||1053.63||3.42||H4 |- | A5*||11.98||718.73||5.01||A5* |- | B5*||12.10||726.10||4.96||B5* |- | C5||13.53||811.88||4.43||C5 |- | D5||14.28||856.59||4.20||D5 |- | E5||18.77||1125.92||3.20||E5 |- | F5||13.74||824.13||4.37||F5 |- | G5||15.84||950.23||3.79||G5 |- | H5||18.66||1119.88||3.21||H5 |- | A6||14.10||845.88||4.26||A6 |- | B6||15.23||913.85||3.94||B6 |- | C6||15.54||932.41||3.86||C6 |- | D6||14.23||853.91||4.22||D6 |- | E6||13.29||797.58||4.51||E6 |- | F6||13.74||824.66||4.37||F6 |- | G6||14.29||857.67||4.20||G6 |- | H6||13.94||836.22||4.31||H6 |- | A7||15.95||956.98||3.76||A7 |- | B7||14.68||880.72||4.09||B7 |- | C7||14.81||888.76||4.05||C7 |- | D7||15.94||956.39||3.76||D7 |- | E7*||6.96||417.31||8.63||E7* |- | F7**||4.17||250.11||14.39||F7** |- | G7||15.68||940.75||3.83||G7 |- | H7*||13.03||781.77||4.60||H7* |- | A8||15.51||930.69||3.87||A8 |- | B8||14.18||850.94||4.23||B8 |- | C8*||12.09||725.59||4.96||C8* |- | D8||11.08||664.88||5.41||D8 |- | E8**||2.68||160.74||||E8** |- | F8**||4.95||297.18||||F8** |- | A9||8.36||501.40||||A9 |- | B9||7.97||478.09||||B9 |- | C9||8.74||524.33||||C9 |- | D9||7.70||461.86||||D9 |- | E9||6.27||376.26||||E9 |- | F9||7.92||474.91||||F9 |- | G9||4.52||270.94||||G9 |- | H9||7.51||450.74||||H9 |} * Note: For the sequencing libraries, NP-BSPP-SR_Ind1-60_May21,2012, I combined samples in well ID A1-D8 (total 60 libraries) * Performed PAGE size selection with 6% TBE 5-well gel (4 gels) [[File:ZhangLab_2 2012-05-28 11hr 15min_PAGE-SS-1.jpg| 300px]] [[File:ZhangLab_2 2012-05-28 11hr 17min_PAGE-SS-2.jpg| 300px]] * Resuspended with H2O, total volume 60ul * Quantified concentration by Qubit dsDNA assay by Alan (based on size 375bp): 83.18nM or 14.4ng/ul * Concentration by PAGE quantification is 33.5ng/ul [[File:ZhangLab_2 2012-05-30 17hr 20min_PQ_NP_BSPP_SR_Ind1-60_May21_2012.jpg| 180px]] * '''Library IDs: NP-BSPP-SR_Ind1-60_May21,2012''', sequenced on '''HiseqMay29''' run at BioGem (Lane: 4-8) ** '''Note: Mapping was performed by Dinh'''. ** '''Raw reads: /media/SeqStore2/120531_SN100_DD/NP_BSPP''' * The rest of libraries (Ind61,62,65-72) were pooled in another library, '''NP_BSPP_SR_HV_Ind61.62.65-80_May31,2012:''' continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-31 * Update: Library IDs: '''NP_BSPP_SR_HV_Ind61.62.65-80_May31,2012''', sequenced on '''HiSeqJun7''' run at Biogem ('''Lane: 8''')
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