User:Karmella Haynes/Notebook/BioBrick cloning/2011/08/06

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08/06/11 ✓ Minipreps: cultures from yesterday; KAH234 (2), KAH231, 232, 228/MV1 ✓ Assembly: KAH235 ✓ Retransformations: DPRE (KAH014), MV1 (pcDNA 3.1+) Minipreps > Check with E/P digests Reagent Volume Expected: 1,2. KAH234 = 1041 Vector frag. = 4072 Insert + 1326... 3,4. KAH231/MV1 = 2914 5,6. KAH232/MV1 = 3106 7,8. KAH228 = 3326 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 KAH235: (1) DPRE/(E/S)/1740 + (2) KAH234/(E/X)/1041 > Digests (Fermentas FD) 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. DPRE (E/S) 1.280 1.91 64.0 2. KAH234 (E/X) 0.465 2.07 23.2 > Ligations Ligation Plate results (lig : neg crtl) 08/07/11 1. DPRE(E/S)/1740, 22 ng + KAH234(E/X)/~4041, 25 ng KAH235 >10:1 (Pick 2) 2. KAH234(E/X)/ 20 ng     1 2 Insert DNA 0.3 --- Vector DNA 1.0 1.0 2x lgn buf (Roche) 5.0 5.0 T4 ligase (NEB) 1.0 1.0 dH2O 2.7 3.0   10 μL 10 μL --> Add to 30 μL DH5α Turbo; plate on 100 μg/mL Amp