User:Karmella Haynes/Notebook/BioBrick cloning/2011/05/24

Project name

Main project page Previous entry      Next entry

05/24/11 ✓ Cultures/minipreps/digests: 2 mL cultures for KAH183(2); KAH184(4) Minipreps > Check with E/P digests Reagent Volume Expected: 1,2. BB 1 = 2383 3-6. BB2 = 2191 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. --> KAH184 not successful. Re-do with higher ratio of insert Assemblies --> Re-build KAH184 KAH184: HPK/(E/S)/516 + KAH182/(E/X)/4869 > Ligations Ligation Plate results (lig : neg crtl) mm/dd/yy 1. HPK(E/S)/516, 15 ng + KAH182(E/X)/4869, 25 ng KAH183 10:1 (Pick 2) 2. KAH182(E/X)/ 25 ng     1 2 3 Insert DNA 3.0 --- Vector DNA 3.5 3.5 2x lgn buf (Roche) 7.5 5.5 T4 ligase (NEB) 1.0 1.0 dH2O --- 3.0   15 μL 15 μL 15 μL ---> Add 15 μL to 40 μL DH5α Turbo