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== Adapter preparation == Assume DNA amount ~100ng, size 400bp MW dA-tailed products = (400bp*607.4 D/bp +157.9 D) = 243.12kD = 243,118 g/mole For 100 ng dA-tailed product = 100ng/243118 g/mole = 4.11E-04 nmole * 20:1 = 0.0082 nmole adapters required. 20uM adapters (ul) required = 0.0082nmoles/(20xE3 nmoles/L * 1E-06L/ul) = 0.0082nmoles/(20E-3 nmoles/ul) = 0.411ul I will prepare total volume of 20uM annealed adapters 10ul {| {{table}} | Stoffel buffer||1.00 |- | PE_t_adapter||2.00 |- | PE_b_adapter||2.00 |- | H2O||5.00 |} * 94C 2min -> 0.2C/sec to 20C -> 4C hold * Dilute annealed adapter with 1x Stoffel buffer 0.41ul to 9.59ul -> anneal adapterr 4.1ul: 1x Stoffelul 95.9 {| {{table}} | 20uM annealed adapter||4.10 |- | 10x Stoffel buffer||10.00 |- | H2O||85.90 |} * Aliquot to column #12: 13ul * After finish adapter ligation, adapter ligated DNA was resuspended in 15ul EB buffer
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