IGEM:University of Chicago/2009/Notebook/Organophosphate Hydrolase/2009/07/09
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Goal: Need to miniprep, quantify, digest/agarose pCTCON2-GFP from... 11:00 - Started 4 hour EBY100 cultures for transfection from single colony tubes 3 x 10 ml, OD660 .203, .166, .194. Aliquotted 12 rxns worth of Zyppy miniprep kits. Annie miniprepped pCTCON2-GFP plasmid (30 ul TE@165.4 ug/ml) Restriction digest to confirm: BAMHI and EcoRI, 1 hour @ 37 degrees C 1 ul 10 x buffer 1ul 10 x BSA 0.5 ul EcoRI 0.5 ul BamHI 1 ul DNA 6 ul H2O 14:40 start incubation -Insert image here- Transformation of pCTCON2-GFP into EBY100 Final OD660: 0.428 Resuspended twice in LiOAc b/c first did it in 500 ml. Heatshock 25 min instead of 20 min. Used herring sperm ssDNA b/c Salmon sperm ssDNA thick like jelly. Provided by Eric: /[A-Z] 6898/? Used 3 ul b/c 5 mg/ml instead of 10 Plated control, pCTCON2- (?), pCTCON2-GFP, 8 ul each. DNAwt: N/A, 9.6 ug(!?), 1.28 ug Tomorrow: Figure out mystery tubes. Repeat restriction digest? (or at least figure out expected fragments) Make glycerol stocks of EBy100; E. coli w/ pCTCON2,E.coli w/pCTCON2-GFP Saturday: check today's transformants check Nora's transformants from yesterday design paraoxon trials |