User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/21

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07/21/11 ✓ Cultures/minipreps: miR-luc 196/MV8 set (9 constructs, 3 each); KAH219, 223, 224/pSB31 (4 each); KAH213, 219/MV1 (3 each) Minipreps miR-luc 196/MV8 set: ApaLI KAH219, 223, 224/pSB31: KpnI/NcoI KAH213, 219/MV1: E/P Reagent Volume Expected: MV8 bands = 1243, 1135, 288 1-3. 186-196/MV8 = 3688*, 2772**, 711 4-6. 184-196/MV8 = 3854*, 2772**, 711 7-9. 187-196/MV8 = 3932*, 2772**, 711 10-12. 188-196/MV8 = 3787*, 2772**, 711 13-15. 189-196/MV8 = 3959*, 2772**, 711 16-18. 190-196/MV8 = 4031*, 2772**, 711 19-21. 191-196/MV8 = 3886*, 2772**, 711 22-24. 183-196/MV8 = 4046*, 2772**, 711 25-27. 192-196/MV8 = 4124*, 2772**, 711 *insert + 234 (vector) + 1429 (196 frag) **last 196 frag + 1655 pSB31 = 4500, 450 28-31. 219/pSB31 = 4500*, 150*, 444, 1735, 532** 32-35. 223/pSB31 = 4500*, 150*, 636, 1190** 36-39. 224/pSB31 = 4500*, 150*, 636, 1490** *pSB31 bands **last frag + 300 MV1 frag. = 4072 40-42. 213/MV1 = 4160 43-45. 219/MV1 = 3748 15 μL/lane; 1% agarose 15 μL/lane; 1% agarose 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 Oligo annealing New BB 1 New BB 2 DNA (oligos, 100 μM) up to 18 μL (3 μL ea.) 10x annealing buffer 2.0 dH2O ---   20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight