User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/30

Project name

Main project page Previous entry      Next entry

11/30/09 ✓ Minipreps: KAH99(2), 100(2), 101(2), 102(2), 103(2), 104(2) ✓ Assemblies: KAH011; KAH93, 94, 95 (RFP miRNA reporters) into pcDNA3.1+puro/ΔCMV (MV2) Minipreps > Check with E/P digests, E/P/ApaLI for KAH99, 100, 101 > ApaLI fragments = 1246, 776, 745, 497 Reagent Vol (1-6) Vol (7-12) Expected: 1,2. KAH99 = 2913 3,4. KAH100 = 3085 5,6. KAH101 = 3157 7,8. KAH102 = 374 9,10. KAH103 = 546 11,12. KAH104 = 540 15 μL/lane; 1% agarose DNA(plasmid) 1.0 2.0 10X buffer 1.5 1.5 EcoRI 1.0 1.0 PstI 1.0 1.0 ApaLI 1.0 --- dH2O 9.5 9.5   15 μL 15 μL --> 37°C/ ~15 min. Assemblies KAH011: NLS-myc-stop (KAH011 dsOligo)/(X/P)/64 ✓ + V0120/(X/P)/3200 ✓ KAH93/MV2: (1) KAH93/(X/P)/4839 + pcDNA3.1+puro/ΔCMV(MV2)/(S/P)/4529 ✓ KAH94/MV2: (2) KAH94/(X/P)/5011 + " KAH95/MV2: (3) KAH95/(X/P)/5083 + " > Digests (Fermentas FD) 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. KAH93 (X/P) 0.440 1.87 22.02 2. KAH94 (X/P) 0.165 2.97 8.24 3. KAH95 (X/P) 0.125 2.56 6.27 > Ligations Ligation Plate results (lig : neg crtl) 12/01/09 1. KAH011(X/P)/64, 0.8 ng + V0120(X/P)/3200, 20 ng KAH011 10:1 (Pick 8) 2. V0120(X/P)/ 20 ng   3. KAH93(X/P)/4839, 43 ng + MV2(S/P)/4529, 20 ng KAH93/MV2 1:1 (re-do) 4. KAH94(X/P)/5011, 44 ng + " KAH94/MV2 1:1 (re-do) 5. KAH95(X/P)/5083, 45 ng + " KAH95/MV2 1:1 (re-do) 6. MV2(S/P)/ 20 ng     1 2 3 4 5 6 Insert DNA 0.5 --- 2.0 5.3 7.0 --- Vector DNA 4.0 4.0 0.5* 0.5* 0.5* 0.5* 2x lgn buf (Roche) 5.5 5.5 8.5 8.5 8.5 8.5 T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 dH2O --- 0.5 5.0 1.7 --- 7.0   11μL 11μL 17μL 17μL 17μL 17μL --> R.T./~10 min; Add 25 μL Z-DH5α --> * Dilute MV2 1:5 w/ dH2O