09/07/ 17 (yy/mm/dd) Experimenter Kashima,Shihoya Note-taker Kashima Title Flow Chart Sample Date Sample NameEnzymepurification KitProductStorage 1.Miniprep.

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09/07/ 17 (yy/mm/dd) Experimenter Kashima,Shihoya Note-taker Kashima Title Flow Chart Sample Date Sample NameEnzymepurification KitProductStorage 1.Miniprep Kit 2.Insert check iGEM DNA parts kits 1-1(2-A-4+2-O-2) ①②③ 6F freezer 7/17 Miniprep Kit, Insert check 1.5ml tube each x1 Miniprep Kit

(Discussion) Cultured sample ( :00 ～ :00 ) ;1-1 ①②③ Centrifuge 4 ℃, × g, 2min Remove the top of layer twice Photo (Fig.1) P1 250ul P2 250ul N3 350ul Centrifuge 4 ℃, × g, 10min Apply the top of layer to column Centrifuge RT, 10sec and remove through Buffer PB 500ul Centrifuge RT, 10sec and remove through Buffer PE 650ul Centrifuge 4 ℃, × g, 1min Exchange tube (remove C 2 H 5 OH) Milli Q 50ul RT 1min Centrifuge 4 ℃, × g, 1min Collect through and measure concentration

Result of Miniprep Kit (Discussion) Sample:conc. (ng/ul) 1-1 ① ② ③ 93.7

Description (Insert check) 37 ℃ incubate ( time: :20 ～ :55 ) (Discussion) Restriction Enzyme Digestion Gel :Agarose Time : 11:05 ～ 11:40 Voltage: 100V Maker: 1Kbp Electrophoresis 123 Sample 1-1 ① 1-1 ② 1-1 ③ 3ul enzyme EcoR1 0.3ul enzyme H2OH2O1ul H buffer5.5ul 6×lodding buffer2ul Total12ul 123 Sample 1-1 ① 1-1 ② 1-1 ③ 3ul enzyme EcoR1 0.3ul enzyme H2OH2O1ul H buffer5.5ul Total10ul

Result of Insert check (Discussion) We checked that ①, ② and ③ colonies have the plasmid incerted 1-1(2-A-4+2-O-2 :4.7kbp)