User:Karmella Haynes/Notebook/BioBrick cloning/2011/06/29

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06/29/11 ✓ Biobrick PCR: SP1A and SP1B (from s.d. mutated SP1AB) ✓ Assemblies: KAH199 (spacer + TK), KAH200 ✓ SD mutagenesis: re-do mutagenesis of PstI site #2 in p65 ✓ Cultures/ minipreps: KAH187-198/MV8 (2); concentration of stock is low Biobrick PCR: SP1A, SP1B > Note: Last step, complete SP1AB mutation, was confirmed on 3/25/11 KAH018: (1) SP1A/(X/P PCR)/540 + V0120/(X/P)/~3200 ✓ KAH019: (2) SP1B/(X/P PCR)/840 + " > PCR --> Template: SP1AB Biobrick --> Primers 1: BB_SP1A f/ BB_SP1A r --> Primers 2: BB_SP1B f/ BB_SP1B r Reagent Volume DNA (plasmid) 1.0 10 μM primers 1.0 2x GoTaq 25.0 dH2O 23.0 --> BioRad DNA Engine, block A 95°C/ 3 min. [95°C/ 30 sec; 55°C/ 30 sec; 72 °C/ 1 min.] x30 72 °C/ 3 min. 4 °C/ ∞ > Zymo clean PCR; elute w/ 25 μL dH2O > Digests (Fermentas FD) Reagent Volume   30 μL/lane, 1% agarose Expected: 2. SP1A = 540, 3. SP1B = 840 PCR DNA 25.0 10x buffer 3.0 XbaI 1.0 PstI 1.0 dH2O ---   30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. SP1A (X/P PCR) 0.912 1.86 45.6 2. SP1B (X/P PCR) 0.614 1.80 30.7 > Ligations Ligation Plate results (lig : neg crtl) 06/30/11 1. SP1A (X/P)/540, 8 ng + V0120(X/P)/~3200, 25 ng SP1A (KAH018) >10:1 (Pick 2) 2. SP1B (X/P)/840, 13 ng + " SP1B (KAH019) >10:1 (Pick 2) 3. V0120(X/P)/ 25 ng     1 2 3 Insert DNA 0.5 0.5 --- Vector DNA 1.0 1.0 1.0 2x lgn buf (Roche) 5.0 5.0 5.0 T4 ligase (NEB) 1.0 1.0 1.0 dH2O 3.0 3.0 3.5   10 μL 10 μL 10 μL --> R.T./ ~15 min.; Add to 30 μL DH5α turbo Assemblies --> Note: DPRE has PstI sites KAH199: (1) Spacer B433/(E/S dp)/65 + (2) HSVtk TATA DDB81/(E/X)/56 KAH200: DPRE KAH014/(E/S)/1740 ✓ + 5xGal4 KMC01/(E/X)/93 ✓ > Digests (Fermentas FD) --> Specific notes Reagent Volume   30 μL/lane, 1% agarose; Lane 1 = DDB81(E/X) 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. > Zymo clean --> Spacer B433 (E/S) insert --> Elute w/ 25 μL dH2O > Measure conc.'s Sample OD260 260/280 ng/μL 2. HSVtk TATA DDB81 (E/X) 1.677 1.87 83.9 --> Dilute 1:2 to get ~42 μg/μL > Dephosphorylation (Roche) --> Phosphatase-treat the Spacer (E/S) insert Reagent Volume DNA (clean digest) up to 17 μL (500 ng) 10x buffer d.p. 2.0 phosphatase 1.0 dH2O ---   20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL > Ligations --> Use max insert volume for ligation #1 (insert includes old vector) Ligation Plate results (lig : neg crtl) 06/30/11 1. Spacer B433(E/S dp)/65, 1 ng + HSVtk TATA DB81(E/X)/~3256, 25 ng KAH199 >10:1 (Pick 2) 2. HSVtk TATA DB81(E/X)/ 25 ng   3. DPRE KAH014(E/S)/1740, 26 ng + 5xGal4 KMC01(E/X)/~3293, 25 ng KAH200 1:1 (Pick 2) 4. 5xGal4 KMC01(E/X)/ 25 ng     1 2 3 4 Insert DNA 3.5 --- 1.0 --- Vector DNA 0.5 0.5 0.5 0.5 2x lgn buf (Roche) 5.0 5.0 5.0 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 dH2O --- 3.5 2.5 3.5   10 μL 10 μL 10 μL 10 μL --> Add 30 μL DH5α Turbo; plate on 100 μg/mL Amp Site-directed Mutagenesis > Stratagene Quick Change mutagenesis kit. p65 (~4 kb): mutate w.t. clone (two PstI sites within single primer); last attempt mutated only the first site (3/25/11) --> Primer mut_p65 1 Reagent p65 w.t. DNA (plasmid, ~100 ng) 1.0 10x buffer 2.5 Quick Solution 0.5 10 μM primer 1 1.0 dNTP mix 1.0 Quick Change enzyme mix 1.0 dH2O 18.0   25 μL --> BioRad PCR (Block A) 95°C/ 1 min. [95°C/ 1 min., 55°C/ 1 min., 65°C/ 8 min (2 min./kb)] x30 65°C/ 1 min. 4°C/ ∞ (6/30/11) > DpnI Digest (gets rid of methylated template DNA) p65 mutation p65 control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR) --> Add 1 μL DpnI enzyme to each sample --> 37°C/ 1 hr. --> Transform 30 μL z-DH5α, use 10 μL each sample; Amp plates 07/01/11 --> Results p65: 2 colonies (1 on neg ctrl.)