Haynes Lab:Notebook/Synthetic Chromatin for Cancer Research/2015/06/15

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6-4-15 David Tze - assemblies: linkers + hPCD Assemblies DT009_pUC57: flyPCD(E/S)/186 + PLflex_pUC57(E/X)/2763 DT0010_pUC57: flyPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930 DT0011_pUC57: flyPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763 DT0012_pUC57: flyPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931 Digests (Fermentas FD) Reagent Rxn1 Expected bands: 1. fshPCD(E/S) = 3200, 186* (*) Bands that were cut out for purification 30 μL/lane, 1% agarose; Ladder Well 1 was punctured so the ladder was put into Well 3 as well for safe measures. DNA (plasmid) 25.0 10x buffer 3.0 EcoRI 1.0 enzyme 2 1.0 dH2O ---   30 μL --> 37°C/ ~30 min. Gel purification Sigma GenElute Gel purification kit Elute & back-elute w/ 25 μL elution sln. Measure conc.'s Sample OD260 260/280 ng/μL 1. flyPCD(E/S) --- --- --- 2. PLflex_pUC57(E/X) --- --- --- 3. PLflex4_pUC57(E/X) --- --- --- 4. PLrigid_pUC57(E/X) --- --- --- 5. PLrigid4_pUC57(E/X) --- --- --- Readings were odd, which is typical for the Sigma gel extraction products. Will use an arbitrary volume of 2.0 insert and 2.0 vector as it has worked before. Good insert : vector ratio should not be difficult to achieve since insert is very short. Linkers were extracted in a previous experiment. Concentrations were taken from that entry as the same linkers were used. Ligations 2:1 ratio calculation: 186 bp insert / ~2800 bp vector * 2 * 50 = 6.64 ng insert DT009_pUC57: fshPCD(E/S)/186 + PLflex_pUC57(E/X)/2763 DT0010_pUC57: fshPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930 DT0011_pUC57: fshPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763 DT0012_pUC57: fshPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931 PLflex_pUC57(E/X)/2763 Note: Used one negative (one vector only) Reagent Rxn1-4 Rxn5 Insert DNA 2.0 --- Vector DNA 2.0 2.0 2x lgn buf (Roche) 5.0 5.0 T4 ligase (NEB) 1.0 1.0 dH2O --- 2.0   10.0 μL 10.0 μL RESULTS Looks successful: ~200 colonies on the ligation plates, 1 colony on the neg. ctrl plate. Picked two colonies from each plate (1-4) for streak library and 5 mL cultures