User:Tk/Notebook/MF-xfm/2008/04/20

MF-xfm

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Transformation results 73 transformants from last week's transformations Most from transformations with low cell density Most from transformations with 10 ul 1M sorbitol added prior to electroporation nothing shows at 14 hours or 24 hours Some colonies visible at 38 hours, more at 48, final at 60 or so New standard transformation protocol culture cells in 45 ml 1161 medium to light yellow/slightly turbid Spin down and resuspend 3x with 45 ml EPB (no glycerol) on ice in chilled centrifuge Resuspend in 5 ml EPB on ice Aliquot 50 ul cells + 1 ul transposon DNA on ice Add 10 ul 1 M sorbitol Electroporate at 1.5 KV in 1 mm cuvette Immediately add 850 ul 1161 medium, place on ice Incubate 1 hour at 30C in the cuvette Plate 300 ul on 15 ug/ml TET plates Hold remainder in the cuvette for several days at 4C Newly diluted transposase Dilute 50 ul transposase (8 ug/ul) into 800 ul transposase storage buffer Store at -20, label 500 ng/ul transposase New transposomes 20 ul transposon DNA (83 ng/ul) 20 ul glycerol (use cut off tip for pipetting) 40 ul diluted transposase incubate 1 hour at 37C hold at -20C New transformations standard protocol use 10 ul water, 5 ul water, no addition, 5 ul, 10 ul, and 15 ul sorbitol (1 M) additions Test for both positive and negative osmotic pressure changes, and narrower range than last time Plate out all of sorbitol added versions (3 plates) Plate out only 1 plate from water and no addition plates, save remainder at 4C Plates labeled with -10, -5, 0, +5, +10, +15 for water and sorbitol additions (ul added) New plates and culture medium, standard protocol Plans for genetics Promoters constitutive heat shock Reporters YFP, mCherry antibiotic cat gene recT recombination gene Test constitutive and heat shock promoters with reporter Test cat gene with constitutive promoter Combine heat shock promoter with recT gene Test recombination efficiency