User:Karmella Haynes/Notebook/BioBrick cloning/2011/08/21

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08/21/11 ✓ Cultures/minipreps: KAH223, 232, 231: pick 8 new colonies from old PCR cloning plates ✓ Assembly: KAH235/V0200 (re-do) ✓ Cultures for QIAGEN Plus midipreps: KAH222, 219, 232, 231/pSB31 35 ml cultures (innoculated from 2 ml cultures) Minipreps > Check with NcoI digests (p65 has two NcoI sites; expected sizes are for fwd orientation) Reagent Volume Expected: Vector fwd = ~5000 rev = >5000 *Last NcoI frag + 141 1-8. KAH223 = 1031*, 636 9-16. KAH232 = 513, 99, 662*, 636 17-24. KAH231 = 513, 99, 662*, 450 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. --> Keep circled samples and also 3 more of each for sequencing tomorrow Assemblies KAH235/V0200: (1) KAH235/(N/S)/2787 + V0200/(N/S dp)/4442 ✓ > Digests (Fermentas FD) --> Re-cut KAH235 (N/S); separation last time was not good Reagent Volume   30 μL/lane, 1% agarose 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. KAH235 (N/S) 0.172 1.73 8.6 > Ligations Ligation Plate results (lig : neg crtl) 08/22/11 1. KAH235(N/S)/2787, 25 ng + V0200(N/S dp)/4442, 20 ng KAH235/V0200 >10:1 (Pick 2) 2. V0200(N/S dp)/ 20 ng     1 2 Insert DNA 3.2 --- Vector DNA 0.8 0.8 2x lgn buf (Roche) 5.0 5.0 T4 ligase (NEB) 1.0 1.0 dH2O --- 3.2   10 μL 10 μL