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=Barcoding DMR330K probes= New primers: {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Sequence Name''' | align="center" style="background:#f0f0f0;"|'''Sequence''' |- | pAP1V61U_w||5β-/5Phos/GGGTCATATCGGTCACTGTU-3β |- | pAP2V6_s||5β-/5Phos/C*A*C*GGGTAGTGTGTATCCTG-3β |} Noi amplified and purified 2x32 reactions of DMR330K-E mixed probes: Tube 1 = 105ng/ul x 210 ul = 22 ug Tube 2 = 89ng/ul x 210 ul = 18.7 ug Lambda Exo: DMR330K amplicons ~20ug, 210 ul Lambda exo buffer (10x), 25 ul Lambda exo 10 ul H2O 5 ul =============================== 250 ul Purified with 2 columns for each tube. Digested Tube 1 with Lambda exo for 1 hour, yield: 79ng/ul x 70 ul = 5.53 ug Digested Tube 2 with Lambda exo for 1 hour, yield: 98ng/ul x 70 ul = 6.86 ug ===Test circularization #1: previous conditions=== Reaction: Lambda digested probes (79ng/ul, 5ug) 64 ul APV6_splint (100uM) 2.2 ul Ampligase buffer (10x) 8 ul H2O 5.8 ul ================================================ 80 ul Program: 95C 30s -> 94.8C 30s -> (-0.2C ) x 199 cycles -> 55C forever (add 9ul Ampligase + 1ul buffer) -> (55C 2 min -> 94C 30s) x 12 cycles -> 55C forever -> 37C 2min (add 2.5 ul ExoI, 2.5 ul Exo III, 1 ul ExoIII buffer, 4 ul H20) -> 37C 1 hr -> 90C 5min -> 4C forever Purified with column, elute in 50ul EB, yield: 1.3ng/ul x 50 = 65 ng TBU-gel [[File:DMR330K-E circularization with lambda exo.jpg]] A - 10 bp ladder (1ul) B - Linear ssDNA probes (78ng) C - Purified circle probes (1.3ng/ulx10ul = 13ng) ===Test circularization #2: Changing the amount of splint.=== *Amount of splint to probes now changed because targets are single-stranded. Reaction: Lambda digested probes, 0.5 ug 6.3 ul (79ng/ul) APV6_splint + H2O (10uM) 8:1 2.2 + 0.5 ul 4:1 1.1 + 1.6 ul 2:1 0.6 + 2.1 ul Ampligase buffer (10x) 1 ul ================================================ 10 ul Program: 95C 30s -> 94.8C 30s -> (-0.2C ) x 199 cycles -> at 59C add 3 ul of (3 ul Ampligase, 1 ul Ampligase buffer, and 6 ul H2O) -> 55C forever -> (55C 2 min -> 94C 30s) x 40 cycles -> 55C forever -> 4C forever *skip ExoI/III Run TBU gel:
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