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=N2 Adaptor Protocol for Library Contruction on 220K captured PGP1-ips and PGP1-fib libraries= *Capture libraries prep: (http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh/NOTES/2010-7-9) *KZ (http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/ExonomeSeq/2010-1-22), only ~300ng captured PCR amplicons required for library construction ===Experiment Plan:=== [[File:PGP1-ips, PGP1-f 220k capture lib quant.jpg|400px]] 1. 10.16ng/ulx20ul=203ng (PGP1-iPS captured at 58C, 44ng 220K probes (30:1), size selected) 3. 12.76ng/ulx20ul=255ng (PGP1-iPS captured at 60C, 44ng 220K probes (30:1), size selected) ------------------------ (A) Combine 1&3 to get 458ng in 40ul PGP1-iPS captured amplicons. 5. 3.22ng/ulx20ul=64ng (PGP1-fib captured at 58C, 44ng 220K probes (30:1), size selected) 6. 1.18ng/ulx20ul=24ng (PGP1-fib captured at 58C, 44ng 220K probes (30:1), size selected) ------------------------ Will not use 5 & 6 7. 10.89ng/ulx20ul=327ng (PGP1-fib captured at 58C, 176ng 220K probes (110:1), size selected) 8. 12.53ng/ulx30ul=376ng (PGP1-fib captured at 60C, 44ng 220K probes (30:1), non-ss) 9. 4.28ng/ulx30ul=129ng (PGP1-fib captured at 60C, 44ng 220K probes (30:1), non-ss) ------------------------ Use 7 (B) and (C) combine 8 & 9 to get 505 ng in 60ul ===Mme I digestion=== 1 units per 1ug phiX 174 DNA 1 phiX = 5 sites/5389bp 1 amplicon = 2 sites/300bp 7.2 units MmeI to digest 1ug capure PCR amplicons => 2.16 units to digest 300ng. (A) 27ul capture amplicon + 4.8ul H2O (300ng) (B) 17ul capture amplicon + 14.8ul H2O (185ng) (C) 31.8ul capture amplicon (268ng) VOL REAGENT Conc Final 31.8 ul DNA + H2O ~300ng 4.0 ul NEBuffer 4 10x 1x 1.2 ul MmeI 2 units/ul 2.4 units 3.0 ul SAM 1mM 75uM -------------------------------------------- 40.0 ul TOTAL Incubate at 37C for 1 hr. Purify with 1 MinElute column each. Yield: (exp. 70%) A. 92ng (31%) | B. 52ng (28%) | C. 118ng (44%) ===Ligation=== Adaptors preparation: 20ul PE_N2_adaptor (100uM) 20ul PE_b_A (100uM) 10ul stoffel buffer (10x) 50ul H2O ---------------------------- 100 ul TOTAL (20uM adaptors) 94C 2min -> 0.2C/sec to 20C -> 4C hold Adapters to ligated product ratio: 20:1 Est length of ligated products: 250bp (after MmeI) + 2x33bp = 316bp MW ligased products = (316bp*607.4 D/bp +157.9 D) = 19.2kD = 19,200 g/mole For 100 ng ligased product = 100ng / 19,200g/mole = 5.21E-3 nmole * 20:1 = 0.1042 nmole adapters required. Adapters (ul) = 0.1042 / (20xE3nmoles/L * 1E-6L/ul) = 0.1042nmoles/ (20E-3 nmoles/ul) = 5.2 ul -----> 5.2 ul of 20uM adapters per 100ng ligased products. Ligation Reaction DNA Adapters QuickLigase 2xQuickLigase Buffer A. 16ul 3.9ul 2ul 21.9ul B. 16ul 2.2ul 1ul 19.2ul C. 16ul 4.9ul 2ul 22.9ul Incubate at RT for 15 min. Purify with 1 MinElute column each, elute with 15ul EB. Yield (inaccurate because high amount of adapters added). ===Amplification=== 1x 4x Reagents 1.0ul --- adapter ligased DNA 2.0ul 8 PCR_F(10uM) 2.0ul 8 PCR_R(10uM) 0.4ul 1.6 SYBR Green 50x 44.6ul 179 H2O 50.0ul 200 Phusion HF, 2xMM -------------------------------- 100 ul each 98C - 30s 98C - 10s 62C - 20s 72C - 17s measure pt 72C - 3s Go to 2, 10x 72C - 2 min 15C forever Amplify for 8 cycles. Purify with 1 QiaQuick column. [[File:DD 2010-08-20 N2 adaptor protocol.jpg|400px]]
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