User:Karmella Haynes/Notebook/BioBrick cloning/2010/08/07

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08/07/10 ✓ Assemblies: KAH160-170 into KAH015 (lentivirus vector) Assemblies KAH160/KAH015: (1) KAH160/(E/S)/1147 + (12) KAH015/(E/X)/8402 KAH161/KAH015: (2) KAH161/(E/S)/1147 + " KAH162/KAH015: (3) KAH162/(E/S)/1147 + " KAH163/KAH015: (4) KAH165/(E/S)/2131 + " KAH164/KAH015: (5) KAH164/(E/S)/2134 + " KAH165/KAH015: (6) KAH165/(E/S)/991 + " KAH166/KAH015: (7) KAH166/(E/S)/991 + " KAH167/KAH015: (8) KAH167/(E/S)/991 + " KAH168/KAH015: (9) KAH168/(E/S)/1955 + " KAH169/KAH015: (10) KAH169/(E/S)/1958 + " KAH170/KAH015: (11) KAH170/(E/S)/955 + " > Digests (Fermentas FD) Reagent Volume   30 μL/lane, 1% agarose DNA (plasmid) 15.0 10x buffer 3.0 enzyme 1 1.0 enzyme 2 1.0 dH2O 10.0   30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. KAH160 (E/S) 0.071 9.24 3.5 2. KAH161 (E/S) 0.158 4.44 7.9 3. KAH162 (E/S) 0.165 2.99 8.3 4. KAH163 (E/S) 0.204 2.83 10.2 5. KAH164 (E/S) 0.161 3.44 8.0 6. KAH165 (E/S) 0.182 2.75 9.1 7. KAH166 (E/S) 0.138 5.11 6.9 8. KAH167 (E/S) 0.156 2.17 7.8 9. KAH168 (E/S) 0.124 2.22 6.2 10. KAH169 (E/S) 0.086 2.05 4.3 11. KAH170 (E/S) 0.122 3.47 6.1 12. KAH015 (E/X) 0.484 2.11 24.2 > Dephosphorylation (Roche) --> KAH015(E/X) Reagent Volume DNA (clean digest) 17.0 (~400 ng) 10x buffer d.p. 2.0 phosphatase 1.0 dH2O ---   20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~20 ng/μL > Ligations Ligation Plate results (lig : neg crtl) 08/08/10 1. KAH160(E/S)/1147, 8 ng + KAH015(E/X)/8402, 30 ng KAH160/KAH015 >10:1 (Pick 1) 2. KAH161(E/S)/1147, 8 ng + " KAH161/KAH015 >10:1 (Pick 1) 3. KAH162(E/S)/1147, 8 ng + " KAH162/KAH015 >10:1 (Pick 1) 4. KAH163(E/S)/2131, 15 ng + " KAH163/KAH015 >10:1 (Pick 1) 5. KAH164(E/S)/2134, 15 ng + " KAH164/KAH015 >10:1 (Pick 1) 6. KAH165(E/S)/991, 7 ng + " KAH165/KAH015 >10:1 (Pick 1) 7. KAH166(E/S)/991, 7 ng + " KAH166/KAH015 >10:1 (Pick 1) 8. KAH167(E/S)/991, 7 ng + " KAH167/KAH015 >10:1 (Pick 1) 9. KAH168(E/S)/1955, 14 ng + " KAH168/KAH015 >10:1 (Pick 1) 10. KAH169(E/S)/1958, 14 ng + " KAH169/KAH015 >10:1 (Pick 1) 11. KAH170(E/S)/955, 7 ng + " KAH170/KAH015 >10:1 (Pick 1) 11. KAH015(E/X), 30 ng + " 0 colonies   1-11 12 Insert DNA 3.5 --- Vector DNA 1.5 1.5 2x lgn buf (Roche) 6.0 6.0 T4 ligase (NEB) 1.0 1.0 dH2O --- 3.5   12μL 12μL --> R.T./~10 min.; Add to 50 μL T-DH5α cells 8/08/10 --> Many colonies on the ligation plates, none on the negative control. Looks great! Pick one colony from each plate; confirm via digest.