User:Tk/Notebook/MF-xfm/2008/04/06

MF-xfm

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Prepare electrocompetent M.florum cells Grow 45 ml culture in 1161 medium, no antibiotic growth is to yellow, light turbid phase Chill culture on ice for 30 minutes while fast cooling centrifuge Spin down 10 min at 9000g remove supernatent, resuspend pellet Add 45 ml ice cold EPB leave on ice 15 minutes Spin down 10 minutes at 9000g remove supernatent, resuspend pellet Add 45 ml ice cold EPB leave on ice 15 minutes Spin down 10 minutes at 9000g remove all supernatent, resuspend pellet Bring pellet to 250 μl volume (200x concentration) Add 25 μl glycerol (10%) leave on ice for 30 minutes Aliquot 5x 50 μl into 1.7 ml eppendorfs, prechilled on ice Flash freeze in dry ice/ethanol Store at -80C until use Make transposomes Add 2 μl 100 ng/μl ME0 PCR DNA Add 4 μl 500 ng/μl transposase Add 2 μl glycerol incubate at 37C for 1 hour hold at -20C indefinitely Made 80 μl transposomes with ME0/PCR/PCR cleanup DNA 118 ng/μl (20 μl) Will test with E. coli transformations Will test with M. florum transformations Electroporation results 1 ng/ul and 100 pg/ul E. coli transformations sparked at 2.5 KV 10 pg/ul and transposome E. coli transformations worked at 2.5 KV Me. florum transformations with transposomes sparked at 2.5 KV and 2.2 KV, functioned at 2.0 KV Outgrowth for 1 hour, plate on LB amp and LB Tet for E. coli, 1161 agar + tet for Me. florum