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===PCR=== Adapter ligated DNA 20 μl PCR_F(10uM) 2 μl PCR_R.N2IndX(10uM) 2 μl 50X SYBR Green I 0.8 μl H2O 25 μl 2X Phusion HF MM 50 μl ----- 100 μl Barcodes 300bp 800bp B.Jap-1 Ind1 Ind1+7 B.Jap-2 Ind2 Ind2+8 B.Jap-3 Ind3 Ind3+9 Ep.G-1 Ind4 Ind4+10 Ep.G-2 Ind5 Ep.G-3 Ind6 Ind6+12 1A Ind1+7 1F Ind2+10(?) 1G Ind3+11 98C 30 ->(98C 10s -> 65C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x4 -> 72C 2min. Took all except BJ2.3/BJ3.3/BJ2.8/BJ3.8 out after 9 cycles. [[Image:2010-09-10-ligation-PCR.png|450px]] Purified 50ul of each amplicon with 50ul AmPure beads, eluted with 40ul H2O Conc(ng/ul) 300bp 800bp B.Jap-1 12 6 B.Jap-2 12 9 B.Jap-3 3 5 Ep.G-1 10 8 Ep.G-2 4 Ep.G-3 7 7 1A 8 1F 5 1G 6 Combine B.Jap1.3, B.Jap2.3, B.Jap3.3, EP1.3, EP2.3, EP3.3 => Lancelet 300bp library B.Jap1.8, B.Jap2.8, B.Jap3.8, EP1.8, EP3.8 => Lancelet 800bp library 1A, 1F, 1G => GM20431 Chr. library Perform size selection, cut out two different bands from the Lancelet 800bp library. Estimate size: Lancelet-300bp: 250-350bp Lancelet-800bp: 900-1000bp Lancelet-600bp: 600-700bp [[Image:2010-09-10-Lancelet-300bp-lib-size-selection.png|300px]] [[Image:2010-09-10-Lancelet-800bp-lib-size-selection.png|350px]] [[Image:2010-09-10-GM20431-chr-lib-size-selection.png|300px]] [[Image:2010-09-15_mutiplexed_libraries.png|400px]]
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