User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/02

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12/02/09 ✓ Minipreps: KAH011(8); sequencing order ✓ Digests: check MV2 ✓ Assembly: Re-do KAH93, 94, 95 in MV2 (longer MV2 digest, dephos) Minipreps > Check via sequencing (Genewiz) Digests > Cloning with MV2 gives high background > Check MV2 w/ EcoRI, PstI and SpeI individually Reagent Volume Expected = 4529 Lanes: 1. uncut (0 enzyme) 2. EcoRI 3. PstI 4. SpeI 15 μL/lane, 1% agarose DNA (plasmid) 1.0 10x buffer 1.5 enzyme 1.0 (0 for control) dH2O 11.5   15 μL --> 37°C/ ~30 min. Note: Looks like all three cut fine. For cloning (E/P), try a longer digest with more enzyme. Also dephos. the cut vector. Assemblies KAH93/MV2: (1) KAH93/(E/P)/4839 + (4) pcDNA3.1+puro/ΔCMV(MV2)/(E/P)/4529 KAH94/MV2: (2) KAH94/(E/P)/5011 + " KAH95/MV2: (3) KAH95/(E/P)/5083 + " > Digests (Fermentas FD) Reagent Volume   DNA (plasmid) 10.0 10x buffer 3.0 EcoRI 2.0 PstI 2.0 dH2O 13.0   30 μL --> 37°C/ ~1 hr.; Zymo clean > Measure conc.'s Sample OD260 260/280 ng/μL 1. MV2(E/P) 1.441 1.85 72.0 > Dephosphorylation (Roche) --> Do 2 rxn's Reagent Volume DNA (clean digest) 7.0 μL (500 ng) 10x buffer d.p. 2.0 phosphatase 1.0 dH2O 10.0   20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL > Ligations Ligation Plate results (lig : neg crtl) 12/03/09 1. KAH93(E/P)/4839, 43 ng + MV2(E/P)/4529, 20 ng KAH93/MV2 10:1 (Pick 2) 2. KAH94(E/P)/5011, 44 ng + " KAH94/MV2 10:1 (Pick 2) 3. KAH95(E/P)/5083, 45 ng + " KAH95/MV2 10:1 (Pick 2) 4. MV2(E/P)/ 20 ng     1 2 3 4 Insert DNA 1.3 2.3 4.2 --- Vector DNA 0.8 0.8 0.8 0.8 2x lgn buf (Roche) 6.0 6.0 6.0 6.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 dH2O 2.9 1.9 --- 4.2   12μL 12μL 12μL 12μL --> R.T./ ~10 min.; Add 25μL Z-DH5α