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

MF-xfm

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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