User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/25

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01/25/11 ✓ Minipreps: KAH160/pSB31 (10) ✓ Cultures & minipreps: KAH165/pSB31 (10); 2 mL ✓ Assemblies: KAH179, 180 (CFP-Targets) into KAH176, 177, 178/MV8 (RFP-Repressor expression vectors); continue later ✓ Cultures: SP1AB 3x mut (24); 2 mL Minipreps > Check with KpnI/NcoI digests --> pSB31 KpnI is located 142 bp upstream of EcoRI cloning site (according to pdf map) --> BioBrick constructs have 2 internal NcoI sites Reagent Volume Expected (approx.): 1,2. KAH170pSB31 2,5 fwd = 5500, 444, 150 rev = 5010, 640, 444 3-12. KAH160 7-16 fwd = 5500, 636, 150 rev = 5010, 640, 636 Bottom 1-10. KAH165 7-16 = 636, 5480 15 μL/lane; 1% agarose KAH165/pSB31 minipreps, NcoI only DNA(plasmid) 2.0 μL 10X buffer 1.5 KpnI 1.0 NcoI 1.0 dH2O 9.5   15 μL --> 37°C/ ~15 min. --> Conclusions: KpnI digestion unclear; successfully identified KAH160/pSB31 clone; continue w/ picking more KAH165/pSB31 clones before sending plasmids in for sequencing. --> ID'ed two promising KAH165/pSB31 clones; send five putatives off for sequencing (1/26/11 Snow emergency: Genewiz not picking up orders until further notice) Assemblies KAH179/176/MV8: (1) KAH179/(E/S)/2941 + (3) KAH176/MV8/(E/X)/7785 KAH180/176/MV8: (2) KAH180/(E/S)/2743 + " KAH179/177/MV8: KAH179/(E/S)/2941 + (4) KAH177/MV8/(E/X)/7978 KAH180/177/MV8: KAH180/(E/S)/2743 + " KAH179/178/MV8: KAH179/(E/S)/2941 + (5) KAH178/MV8/(E/X)/7989 KAH180/178/MV8: KAH180/(E/S)/2743 + " > Digests (Fermentas FD) --> Note: Re-digested KAH179 w/ E/S/ApaLI Reagent Volume   30 μL/lane, 1% agarose 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. > Measure conc.'s Sample OD260 260/280 ng/μL 1. KAH179 (E/S) 0.167 2.32 8.4 2. KAH180 (E/S) 0.109 3.19 5.5 3. KAH176/MV8 (E/X) 0.407 2.02 20.3 4. KAH177/MV8 (E/X) 0.441 2.22 22.0 5. KAH178/MV8 (E/X) 0.164 2.69 8.2 --> Continue with ligation later