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===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).
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