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

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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 &mu;l volume (200x concentration)
 * Add 25 &mu;l glycerol (10%)
 * leave on ice for 30 minutes
 * Aliquot 5x 50 &mu;l into 1.7 ml eppendorfs, prechilled on ice
 * Flash freeze in dry ice/ethanol
 * Store at -80C until use

Make transposomes

 * Add 2 &mu;l 100 ng/&mu;l ME0 PCR DNA
 * Add 4 &mu;l 500 ng/&mu;l transposase
 * Add 2 &mu;l glycerol
 * incubate at 37C for 1 hour
 * hold at -20C indefinitely
 * Made 80 &mu;l transposomes with ME0/PCR/PCR cleanup DNA 118 ng/&mu;l (20 &mu;l)
 * 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


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