User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/24

Karmella's BioBrick Cloning

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03/24/13 Assembly do-over: used plain LB agar by mistake last time; use new LB Amp plates Ligations Ligation Plate results (lig : neg crtl) 03/21/13 1. A1 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng MV9 ##:## 2. A1 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng MV9 ##:## 3. A1 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng MV9 ##:## 4. MV9(X dp)/ 25 ng   5. MV9(X no dp)/ 25 ng + Behzad's T4 ligase ... 6. MV9(X no dp)/ 25 ng     1 2 3 4 9 10 Insert DNA 0.5 1.0 2.0 --- --- --- Vector DNA 1.0 1.0 1.0 1.0 0.3 0.3 2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 --- dH2O 2.5 2.0 1.0 3.0 3.7 4.7   10 μL 10 μL 10 μL 10 μL 10 μL 10 μL Pre-warm agar-amp plates with lids closed Incubate ligations for 30 min./ room temp Add 30 μL DH5α; 5 min/ ice Plate on 100 μg/mL amp; incubate at 37°C