User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/22

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01/22/11 ✓ Re-do ligation: SP1AB w.t./V0120 ✓ Miniprep: pSB31 (2) ✓ Assemblies: KAH160, 165, 170 into pSB31 Re-do ligations from 01/21/11 > Dephosphorylation (Roche) V0120 X/S vector 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 Ligation Plate results (lig : neg crtl) 01/23/11 1. SP1AB w.t.(X/S)/1617, 25 ng + V0120(X/S)/~3200, 25 ng SP1AB w.t./V0120 5:1 (Pick 6) 2. V0120(X/S)/ 25 ng     1 2 Insert DNA 3.0 --- Vector DNA 1.0 1.0 2x lgn buf (Roche) 5.0 5.0 T4 ligase (NEB) 1.0 1.0 dH2O --- 3.0   10 μL 10 μL --> ~1 hr./ r.t.; Add to 30 μL DH5α turbo Assemblies KAH160/pSB31: KAH169/(E/S blunt)/1147 + pSB31/(E blunt)/5127 KAH165/pSB31: KAH165/(E/S blunt)/991 + " KAH170/pSB31: KAH170/(E/S blunt)/955 + " > Digests (Fermentas FD) --> Digest pSB31 vector w/ EcoRI; 2 rxns --> Do not gel purify Reagent Volume   DNA (plasmid) 20.0 10x buffer 3.0 EcoRI 1.0 dH2O 5.0   30 μL --> 37°C/ ~30 min. --> Zymo clean EcoRI cut vector; elute w/ 25 μL dH2O > Blunting (NEB Quick Blunting kit) --> Blunt all DNA (vectors and inserts) Reagent Volume DNA (clean digest) up to 19 μL 10x blunting buffer 2.5 1 mM dNTP mix 2.5 Blunt Enzyme mix 1.0 dH2O ---   25 μL --> R.T./ 15 min.; 70°C/ 10 min. KAH160 (max. vol.) [final] = 2.7 ng/μL KAH165 (max. vol.) [final] = 6.9 ng/μL JDS170 (max. vol.) [final] = 4.6 ng/μL pSB31 (max. vol., measure after cleaning) --> Clean vector DNA using Zymo DNA Clean & Concentrator; elute w/ 20 μL dH2O > Measure concentration Sample OD260 260/280 ng/μL 1. pSB31 (E blunt) 2.194 1.83 109.7 > Dephosphorylation (Roche) --> pSB31 (E blunt); 3 rxns Reagent Volume DNA (clean) 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 Ligation Plate results (lig : neg crtl) 01/23/11 1. KAH160(E/S blunt)/1147, 11 ng + pSB31(E blunt dp)/5127, 25 ng KAH160/pSB31 5:1 (Pick 6) 1. KAH160(E/S blunt)/991, 10 ng + " KAH160/pSB31 5:1 (Pick 6) 1. KAH160(E/S blunt)/955, 9 ng + " KAH160/pSB31 5:1 (Pick 6) 2. pSB31(E blunt dp)/ 25 ng     1 2 3 4 Insert DNA 3.0 3.0 3.0 --- Vector DNA 1.0 1.0 1.0 1.0 2x lgn buf (Roche) 5.0 5.0 5.0 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 dH2O --- --- --- 3.0   10 μL 10 μL 10 μL 10 μL --> ~1 hr./ r.t.; Add to 30 μL DH5α turbo