User:Karmella Haynes/Notebook/BioBrick cloning/2010/02/02

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02/02/10 ✓ Colony PCR: from EF-1α site directed mutagenesis plates, PCR w/ BioBrick primers ✓ Assembly: EF-1α into V0120 Colony PCR & BioBricking > Pick colonies from site directed mutagenesis plates from 2/01 (6 each) > Use Forward EF1a/ Reverse EF1a (contain Xba and Pst ends) > Cut products with X/P (PstI leaves desired mutants in tact and cuts non-mutated fragments) > Isolate 1184 bp band for cloning PCR 1-6. Forward strand mutagenesis clones 7-12. Reverse strand mutagenesis clones 13. EF-1α (original template plasmid DNA) Reagent Volume Colony pick 0.0 10 μM primer mix 1.0 2x GoTaq Green 12.5 dH2O 11.5   25 μL --> BioRad 96-well 95°C/ 10 min. [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 1 min.] x30 72°C/ 3 min. 4°C/ ∞ --> Zymo clean & concentrate; elute w/ 20 μL dH2O Digest --> Digest samples w/ XbaI/PstI Reagent Volume Expected: 1-6. Fwd strand mut clones = 1184 7-12. Rev strand mut. clones = 1184 13. EF-1α PCR (uncut) 25 μL/ lane; (#13, 10 μL); 1% agarose DNA(clean PCR) 20.0 10x buffer 2.5 XbaI 1.0 PstI 1.0 dH2O 0.5   25 μL --> 37°C/ 30 min. --> Gel purify for Assemblies Assemblies EF-1α(1): ✓ EF-1α PCR 1/(X/P)/1184 + ✓ V0120/(X/P)/3200 EF-1α(2): ✓ EF-1α PCR 2/(X/P)/1184 + " EF-1α(3): ✓ EF-1α PCR 3/(X/P)/1184 + " > Measure conc.'s Sample OD260 260/280 ng/μL 1. EF-1α PCR 1(X/P) 0.191 1.81 9.5 2. EF-1α PCR 2(X/P) 0.143 1.74 7.2 3. EF-1α PCR 3(X/P) 0.229 1.70 11.4 > Ligations Ligation Plate results (lig : neg crtl) mm/dd/yy 1. EF-1α PCR 1(X/P)/1184, 11 ng + V0120(X/P)/3200, 15 ng EF-1α-1 0 colonies 2. EF-1α PCR 2(X/P)/1184, 11 ng + " EF-1α-2 1 colony (Pick 1) 3. EF-1α PCR 3(X/P)/1184, 11 ng + " EF-1α-3 5 colonies (Pick 3) 2. V0120(X/P)/ 15 ng 0 colonies   1 2 3 4 Insert DNA 2.5 2.5 2.5 --- Vector DNA 1.5 1.5 1.5 1.5 2x lgn buf (Roche) 5.0 5.0 5.0 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 dH2O --- --- --- 2.5   10μL 10μL 10μL 10μL --> R.T./ ~10 min.; Add 25 μL z-DH5α