User:Karmella Haynes/Notebook/BioBrick cloning/2012/07/27

Karmella's BioBrick Cloning

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07/27/12 Assembly: clone linker peptides into V0120 DNA sequencing order: order M13F primer sequencing fro FCt2 plasmid online (DNASU) Assemblies > Repeat cloning from 6-27-12 PLrigid/V0120: ✓ PLrigid/(X/P)/60 + V0120/(X/P)/~3200 PLrigid4/V0120: ✓ PLrigid4/(X/P)/240 + " PLflex/V0120: ✓ PLflex/(X/P)/60 + " PLflex4/V0120: ✓ PLflex4/(X/P)/240 + " > Digests (Fermentas FD) --> Digest V0120 backbone only. Inserts are already done. Reagent Volume   DNA (plasmid) 20.0 10x buffer 3.0 XbaI 1.0 PstI 1.0 dH2O 5.0   30 μL --> 37°C/ ~30 min. ---> Zymo clean; elute w/ 15 μL dH2O > Measure conc.'s Sample OD260 260/280 ng/μL 1. V0120 (X/P) 0.009 2.049 8.9 > Ligations --> 60 bp inserts = 1/50 total mass; 240 bp inserts are 1/12 of total mass; multiply ng used accordingly --> Use V0120 from ccdB --> Use dephos insert only as neg ctrls. Ligation Plate results (lig : neg crtl) 07/28/12 1. PLrigid(X/P)/60, 1 ng (x50) + V0120(X/P)/3200, 25 ng PLrigid/V0120 1:1 (try again) 2. PLrigid(X/P)/60, 1 ng (x50) + no vector ~10 3. PLrigid4(X/P)/240, 4 ng (x12) + V0120(X/P)/3200, 25 ng PLrigid4/V0120 1:1 (try again) 4. PLrigid4(X/P)/240, 4 ng (x12) + no vector ~10 5. PLflex(X/P)/60, 1 ng (x50) + V0120(X/P)/3200, 25 ng PLflex/V0120 1:1 (try again) 6. PLflex(X/P)/60, 1 ng (x50) + no insert ~10 7. PLflex4(X/P)/240, 4 ng (x12) + V0120(X/P)/3200, 25 ng PLrigid4/V0120 1:1 (try again) 8. PLflex4(X/P)/240, 4 ng (x12) + no insert ~10   1 2 3 4 5 6 7 8 Insert DNA 2.2 2.2 2.2 2.2 2.2 2.2 2.2 2.2 Vector DNA 2.8 --- 2.8 --- 2.8 --- 2.8 --- 2x lgn buf (Roche) 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 dH2O --- 2.8 --- 2.8 --- 2.8 --- 2.8   12 μL 12 μL 12 μL 12 μL 12 μL 12 μL 12 μL 12 μL --> Add 30 μL DH5α Turbo cells; plate on 100 μg/mL Amp