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==MmeI Digestion== [[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. *Reamplified (A) for 10 cycles with NH2 primers: 109.8ng/ul x 35ul VOL REAGENT Conc (A) (B) (C) (D) (E) 31.8 ul DNA + H2O 3ul 3ul 6ul 6ul 3ul 4.0 ul NEBuffer 4 10x --- ul MmeI 2 units/ul 1.2ul(1x) 2.4ul(2x) 2.4ul(1x) 3.6ul(1.5x) 3.6ul(2x) 3.0 ul SAM 1mM -------------------------------------------- 40.0 ul TOTAL Incubate at 37C for 1 hr. Purify with 1 MinElute column each. Yield: (B) 14.4ng/ulx12.5ul=60%, (E) 16.0ng/ulx13.0ul=69% ===Expected Lengths=== 1. Digested products, (remove linkers (25bp) + 18/20bp) = 300-43-45 = 212bp 2. Adapters ligased products: digested product(212bp)+36(top)+36(bottom) = 284bp 3. Amplified with solexa tail: digested product(212bp)+64(left)+60(right) = 336bp ===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''' Previously calculated 6.6ul per 100ng digested product (too much!) Est length of digested products: 214bp (after MmeI) MW digested products = (212bp*607.4 D/bp +157.9 D) = 128.927kD = 128,927 g/mole For 100 ng digested product = 100ng / 128,927g/mole = 7.76E-4 nmole * 20:1 = 0.02 nmole adapters required. Adapters (ul) = 0.02nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.02nmoles/ (20E-3 nmoles/ul) = 0.77 ul -----> 0.77 ul of 20uM adapters per 100ng digested products. Added 100ng from (B) and (E) but actual digested product ~50% or 50ng. Ligation Reaction DNA Adapters QuickLigase 2xQuickLigase Buffer B. 7.20ul(100ng) 3.3ul 2ul 12.50ul E. 6.25ul(100ng) 3.3ul 2ul 11.55ul Incubate at RT for 15 min. Purify with 1 MinElute column each, elute with 15ul EB. ===Amplification=== 1x Reagents 10.0ul adapter ligased DNA 2.0ul PCR_F(10uM) 2.0ul PCR_R(10uM) 0.4ul SYBR Green 50x 35.6ul H2O 50.0ul Phusion HF, 2xMM -------------------------------- 100 ul each 98C - 30s 98C - 10s 62C - 20s 70C - 30s Go to 2, 7x 72C - 2 min 15C forever Amplify for 8 cycles. Purify with 1 QiaQuick column.
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