Haynes Lab:Notebook/Synthetic Chromatin for Cancer Research/2015/07/10

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6-24-15 Assemblies: linkers/hPCD construct & PcTF (KAH 126) Assemblies DT013_V0120: DT001(E/S)/252 + PcTF_V0120(E/X)/5080 DT014_V0120: DT002(E/S)/432 + PcTF_V0120(E/X)/5080 DT015_V0120: DT003(E/S)/252 + PcTF_V0120(E/X)/5080 DT017_V0120: DT004(E/S)/432 + PcTF_V0120(E/X)/5080 Digests (Fermentas FD) Reagent Rxn1 Expected bands: 1. PcTF(E/X) = 3200, 186* (*) Bands that were cut out for purification File:6.24.15.jpg 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. DT001(E/S) --- --- --- 2. DT002(E/S) --- --- --- 3. DT003(E/S) --- --- --- 4. DT004(E/S) --- --- --- 5. PcTF_V0120(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. Ligations DT013_V0120: DT001(E/S)/252 + PcTF_V0120(E/X)/5080 DT014_V0120: DT002(E/S)/432 + PcTF_V0120(E/X)/5080 DT015_V0120: DT003(E/S)/252 + PcTF_V0120(E/X)/5080 DT017_V0120: DT004(E/S)/432 + PcTF_V0120(E/X)/5080 PcTF_V0120(E/X)/5080 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