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===4. Annealing of guide oligo, fill in with Stoffel, and Ampligase=== * Set up as in capture reaction but with higher annealing temperature * Use 2:1 ratio of guide oligos TaqαI digested ssDNA probes : 1ug 1 ug of ssDNA probes (150 nt, MW = 45,634 g/moles) = 2.191x10^-11 moles TaqαI_RandBar_guide : - Use 0.109 ul of 100uM Insert_guide oligo per 1 ug of digested probes Stoffel (10U/ul): One unit per 10nmoles dNTPs to incorporate per 30 min in 10 min incubation at 74C 0.0219nmolesx44nt = 0.9636nmoles dNTPs to incorporate Reaction will cycle to 74C for a total of 30 min Assumes One unit per 40nmoles, thus ~0.5 Units Stoffel per 1 ug of digested ssDNA probes. dNTP (10mM = 10nmoles/ul): Optimal is 10x the amount required for 100% of targets. 0.0219nmolesx44nt = 0.9636 nmoles dNTPs to incorporate 100*0.9636nmoles/10nmoles/ul = 9.636ul - For 100:1 guide to probes ratio, use 10 ul of 10mM dNTP per 1 ug of digested ssDNA probes Ampligase (5U/ul): One unit per 50% of cos site of 1 ug lambda DNA in 1 minute at 45C - Use 1 unit Ampligase buffer (10x): Use 1x Reaction Mix: SLN for ~ 5 ng of digested product Use 0.5nmoles of dNTP = (1nmoles/ul)x 0.5 ul 0.5 ul dNTP (1 mM) 1.0 ul Stoffel (10U/ul) 0.5 ul Ampligase (5U/ul) 0.5 ul Ampligase buffer (10x) 2.5 ul H2O --------------------------- 5.0 ul total Program: Program C: 95C 30S -> 94.8C for 30s, -0.2C per cycle x 184 cycles -> 58C hold -> add SLN ->(58C 10min -> 94C 30s)x11 -> 58C forever -> 37C 1 min -> Add 1 ul ExoI and ExoIII each -> 37C 45 min -> 90C 10 min-> 4C hold
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