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===Protocol summary from Gosta=== Lab protocol for Tn5 prep 2014-03-27 Gösta Winberg and Rickard Sandberg added by us: *'''Tues:''' Transform C3013 bacterias and grow up plate overnight (amp selection) *'''Wed:''' Inoculate (several, like five 5 mL cultures with same clone in the morning. A600 of .9 takes a long time to reach) 5 mL culture and grow up overnight at 30C (amp selection). *'''Thurs:''' add all cultures to 1L culture, grow up, express protein, centrifuge, freeze -80 O/N *'''Fri:''' sonicate pellet debris, PEI precip, chitin column binding/wash, DTT cleavage and leave over weekend at 4C. *'''Mon:''' dialysis with 2X dialysis buffer, A280 molarity calculation. dialysis with storage buffer, and tn5 fragmentation assay '''Thursday 27/3:''' *Thaw cell pellet and sonic in 80 mL HEGX, pellet debris. *PEI precipitation, pellet PEI-DNA precipitate. *Supernate to 7 mL Chitin column at 0.4 mL/min = 3,5 h. *Wash HEGX, 100 mL at 4 mL/min =gravity flow, 25 min *Last wash degassed 2 col. vol. *Add 8 mL degassed HEGX containing 200 nmoles of MEDS oligo (mixed 1030+1031 ds oligos). (Better to use at least 2x molar excess MEDS oligo) *A 7 mL column can bind max 200 nmoles of Tn5 fusion protein (28 nmoles/mL). *Leave O/N at RT for binding to the Tn5 protein. '''Friday 28/3:''' *Move column to +4. Wash with HEGX to remove excess unbound oligo. *Add 20 mL HEGX, 100 mM DTT. Drain out 15 mL , then close the valve. *Leave column at +4 over weekend. '''Mo 31/3:''' *Elute 4 fractions of 20 drops, then 12 x 40 drop fractions. *Test with BioRad protein assay: 25 uL + 1 uL of column fraction. *Pool most active fractions (most intense blue color). *Dialyze vs 2 x 1L Tn5 dialysis buffer at 4 C. '''Tue 1/4:''' *Read OD280 vs dialysis buffer as blank. *Calculate Tn5 molarity (only approximately since the oligo influences the readings. Run Tn5 assay with 50 ng *High MW DNA as substrate, Run 1% agarose gel to check activity.
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