User:Karmella Haynes/Notebook/BioBrick cloning/2010/06/01

Project name

Main project page Previous entry      Next entry

06/01/10 ✓ Assemblies: make MV8 and MV3 Assemblies MV8: MV1/(BsaBI/BstBI)/4605 + Hygro PCR/(BsaBI/BstBI)/1023 MV3: MV7/(BsaBI/BstBI)/3919 + " > Digests (NEB) --> Specific notes Reagent Volume   30 μL/lane, 1% agarose DNA (plasmid) 15.0 10x buffer 4 3.0 BsaBI 1.0 BstBI 1.0 dH2O 10.0   --> 60°C/ 1 hr.; 65°C/ 1 hr. > Measure conc.'s Sample OD260 260/280 ng/μL 1. MV1 (BsaBI/BstBI) 0.636 1.80 31.8 2. MV7 (BsaBI/BstBI) 1.061 1.77 53.0 > Ligations Ligation Plate results (lig : neg crtl) 06/03/10 1. Hygro PCR(B/B)/1023, 11 ng + MV1(B/B)/4605, 25 ng MV8 1:1 (redo) 2. MV1(B/B)/ 25 ng ~30 colonies 3. Hygro PCR(B/B)/1023, 13 ng + MV7(B/B)/3919, 25 ng MV3 1:1 (redo) 4. MV7(B/B)/ 25 ng ~30 colonies   1 2 3 4 Insert DNA 1.2 --- 1.5 --- Vector DNA 0.8 0.8 0.5 0.5 2x lgn buf (Roche) 5.0 5.0 5.0 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 dH2O 2.0 3.2 2.0 3.5   10 μL 10 μL 10 μL 10 μL --> R.T./ ~10 min.; Add 50 μL Turbo DH5α