User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/02

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07/02/11 Cultures/ minipreps: KAH200 (4) Minipreps > Check with E/P digests Reagent Volume Expected: 1-4. KAH200 = 226 Hover name 15 μL/lane; 1% agarose DNA(plasmid) 2.0 μL 10X buffer 1.5 EcoRI 1.0 PstI 1.0 dH2O 9.5   15 μL --> 37°C/ ~15 min. Assemblies BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size > Digests (Fermentas FD) --> Specific notes Reagent Volume   DNA (plasmid) up to 25 μL 10x buffer 3.0 enzyme 1 1.0 enzyme 2 1.0 dH2O ---   30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. Digested part (a/b) --- --- --- 2. Digested part (c/d) --- --- --- > Dephosphorylation (Roche) Reagent Volume DNA (clean digest) up to 17 μL (500 ng) 10x buffer d.p. 2.0 phosphatase 1.0 dH2O ---   20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL > Ligations Ligation Plate results (lig : neg crtl) mm/dd/yy 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #) 2. vector(c/d)/ ## ng     1 2 Insert DNA ### --- Vector DNA ### ### 2x lgn buf (Roche) ### ### T4 ligase (NEB) 1.0 1.0 dH2O ### ###   # μL # μL