User:Karmella Haynes/Notebook/BioBrick cloning/2010/04/14

Project name

Main project page Previous entry      Next entry

04/14/10 ✓ Minipreps: KAH96, 142, 146, 147/V0201 (12 total); KAH154-159 (12 total) ✓ Assemblies: KAH154-159/MV2 ✓ Cultures: KAH142(6), 146(6), 147/V0201(8) ✓ Plasmid transformations: MV2 and V0201 Minipreps > Check KAH96, 142, 146, 147/V0201 with XbaI digest (add 641 to expected insert size) > Check KAH154-159 with E/P digest Reagent Volume Expected: V0201 band = 4516 Insert band = BB size + 641 bp 1-3. KAH96/V0201 = 3466 4,5. KAH142/V0201 = 5212 6-8. KAH146/V0201 = 7027 9-12. KAH147/V0201 = 8773 --- 13,14. KAH154 = 1724 15,16. KAH155 = 1724 17,18. KAH156 = 1724 19,20. KAH157 = 1724 21,22. KAH158 = 2688 23,24. KAH159 = 2691 15 μL/lane; 1% agarose DNA(plasmid) 2.0 μL 10X buffer 1.5 EcoRI 1.0 PstI 1.0 dH2O 9.5   15 μL           --> 37°C/ ~15 min. --> Pick more colonies for KAH142, 146, 147/V0201 Assemblies KAH154/MV2: (1) KAH154/(E/P)/1724 + (7) MV2/(E/P)/4529 KAH155/MV2: (2) KAH155/(E/P)/1724 + " KAH156/MV2: (3) KAH156/(E/P)/1724 + " KAH157/MV2: (4) KAH157/(E/P)/1724 + " KAH158/MV2: (5) KAH158/(E/P)/2688 + " KAH159/MV2: (6) KAH159/(E/P)/2691 + " > Digests (Fermentas FD) --> Triple digest KAH158, 159 w/ E/P/ScaI --> Digest for 1 hour to ensure complete digestion Reagent 1-4, 7 5, 6 DNA (plasmid) up to 25 μL up to 25 μL 10x buffer 3.0 3.0 enzyme 1 1.0 1.0 enzyme 2 1.0 1.0 ScaI --- 1.0 dH2O --- ---   30 μL 30 μL --> 37°C/ ~1 hr. > Measure conc.'s Sample OD260 260/280 ng/μL 1. KAH154 (E/P) 0.776 1.86 38.8 2. KAH155 (E/P) 0.869 1.88 43.4 3. KAH156 (E/P) 0.873 1.88 43.6 4. KAH157 (E/P) 0.875 1.90 43.7 5. KAH158 (E/P) 0.753 1.84 37.6 6. KAH159 (E/P) 0.854 1.93 42.7 7. MV2 (E/P) 0.467 2.10 23.4 > Dephosphorylation (Roche) MV2 (E/P) Reagent Volume DNA (clean digest) 21.5 (~500 ng) 10x buffer d.p. 2.5 phosphatase 1.0 dH2O ---   25 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 20 ng/μL > Ligations Ligation Plate results (lig : neg crtl) mm/dd/yy 1. KAH154(E/P)/1724, 15 ng + MV2(E/P)/4529, 20 ng KAH154/MV2 #:1 (Pick #) 2. KAH155(E/P)/1724, 15 ng + " KAH155/MV2 #:1 (Pick #) 3. KAH156(E/P)/1724, 15 ng + " KAH156/MV2 #:1 (Pick #) 4. KAH157(E/P)/1724, 15 ng + " KAH157/MV2 #:1 (Pick #) 5. KAH158(E/P)/2688, 24 ng + " KAH158/MV2 #:1 (Pick #) 5. KAH159(E/P)/2688, 24 ng + " KAH159/MV2 #:1 (Pick #) 2. MV2(E/P)/ 20 ng     1 2 3 4 5 6 7 Insert DNA 0.5 0.5 0.5 0.5 1.0 1.0 --- Vector DNA 1.0 1.0 1.0 1.0 1.0 1.0 1.0 2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 dH2O 2.5 2.5 2.5 2.5 2.0 2.0 3.0   10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL --> R.T./ ~10 min.; Add 28 μL Turbo DH5α (T-DH5α); ice/ 5 min. --> plate on warm LB, 100 μg/mL Amp; R.T. overnight; 37 °C next day for ~3 hours