User:Tk/Notebook/MF-xfm/2008/04/25

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


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Transformations

 * Transform 400 ul sample frozen at -80
 * Thaw on ice
 * Add 16 ul transposome (4/24)
 * Add 80 ul 1 M sorbitol
 * Dispense 4x into 1 mm chilled cuvettes
 * Hold on ice 10 min
 * Electroporate at 1.5 KV
 * Immediately add 1 ml 1161 medium
 * Hold on ice 10 min
 * Incubate in the cuvette for 50 minutes at 30C
 * Plate out 330 ul onto 12x 1161 + tet plates

More transposon construction

 * Grow pTT01 plasmid (10 ml overnight culture + 500 ml LB/Kan in a shake flask)
 * Harvest in conical centrifuge tubes after 9 hour growth
 * Discard supernatent, freeze pellet

PCR transposon construction

 * 100 ul PCR reaction
 * 50 ul Phusion m/m
 * 47 ul water
 * 3 ul ME0 primer
 * cycle 98/30s (98/15s 55/15s 68/1:15 min)x36


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