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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