User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/07

AU CHEM-570 Lab Prep

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Objective Try electroporation Re-ligate with different I:V Bench work Glycerol stock of ER2566 250μL 60% sterile glycerol + 750μL ER2566 culture from yesterday → 80 Electrocompetent DH10B This was carried out with the assistance of User:Daniel Catt Inoculate 50 mL LB with 1 mL O/N E. coli DH10B culture from yesterday t=0 OD600=0.08 t~1.5h OD600~0.2 t~2h OD600~0.6 → stop growth incubate culture ~10' on ice w/ swirling spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C Sorvall RC6+ and SH-3000 rotor supernatent discard pellet resuspend in 100 mL cold sterile H2O spin in 2x50 mL falcon tube 20' @ 4500 RPM @ 4°C supernatent discard pellet resuspend in 45 mL cold sterile H2O (should be 50 mL, but not enough sterile water) spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C supernatent discard pellet resuspend in 10 mL cold sterile 10% glycerol spin in 15 mL falcon tube 10' @ 3800 RPM @ 4°C supernatent discard pellet resuspend in 200 μL cold sterile 10% glycerol aliquot into 5 x 50μL (*Kathryn Muratore 15:34, 8 July 2011 (EDT): I meant to freeze the 5th unused aliquot, but did not) Transformation by electroporation Used David Carlini's Bio-Rad electroporator in Biology 0.1 cm cuvettes 50μL DH10B + 20μL V(His)+I ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week] 50μL DH10B + 20μL V(His) neg ctrl ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week] 50μL DH10B + 20μL V+I ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week] 50μL DH10B + 20μL V neg ctrl ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week] 1.5 kV, t = 3.6-4.0 ms add 450μL SOC → 1.5h @ 37°C w/ shaking (should be 45') plate 100μL of 1:1 for all samples on LBAmp100 plate 100μL of 1:100 (diluted with SOC) for V(His)+I and V+I on LBAmp100 → 37°C O/N Re-plate transformants spread remaining 450μL of NovaBlue transformations from yesterday → 37°C O/N Ligation 8.5μL ddV(His) from last week + 4μL ddI from last week + 2μL Ligase buffer + 4.5μL sterile H2O + 1μL Muratore:Materials/DNA ligase 8.5μL ddV from last week + 4μL ddI from last week + 2μL Ligase buffer + 4.5μL sterile H2O + 1μL Muratore:Materials/DNA ligase → 16°C O/N Results No transformants on yesterday's plates