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===IV. Chitin column=== '''Without transposons''' *1. Wash '''Chitin resin at RT with RT HEGX'''. take 15 mL of resin, let settle or spin down in 50 mL conical at 100xg for <1 minute with slow setting 5 for deceleration of centrifuge. Wash 3X with 1X HEGX, remove supernatent with aspirator. *2. Add supernatant from PEI to washed chitin resin in conical. Lightly mix on shaker for 3.5 hours '''at 4C''' *3. Wash bound chitin beads 5X with '''RT 1X HEGX at RT''' in 50 mL conical. spin down at 100 xg for <1 minute with setting of 5 for deceleration of centrifuge, remove supernatent with aspirator. *4. Add solution from conical to chromatography column, after draining put upper bed support on top, and wet with buffer. *5. Wash with 20-30 volumes (200-300 ml) HEGX at gravity flow (2-4 ml/min) '''at RT''' OR wash overnight at 0.2ml/min at 4C *6. Add 30 ml Cleavage buffer +100 mM DTT to column. **a. 462.75 mg DTT in 30 mL (2.5 mL 1M DTT) **b. 15 mL 2X HEGX **c. 14.5 mL H20 **d. 30 mL Final volume *7. Run out until ~7-8 mL (not including beads) cleavage buffer is in column, stop flow and '''leave at 4C''' for at least 36 hours (over weekend) '''Did for 63 hours last time''' *8. Elute first fraction of ~5 drops to discard (dead volume, no protein). Then elute ~10 fractions of 10 drops (~500 uL), then 12x20 (~1 mL) drop fractions. *9. (optional) Leave the column in the remaining Cleavage buffer +100 mM DTT for 24h. (Checked: Up to 30% may be in elute2). '''With Transposons''' *1. Add supernatant from PEI precipitation to 10ml Chitin column at 0.4ml/min in HEGX (~3.5 hours) at 4C. *2. Wash with 10-20 volumes HEGX, (100-200 mL) at gravity flow at 4C (~4 mL a minute = 25 mins) *3. Repeat wash with 2 column volumes (200 mL) degassed HEGX at gravity flow at 4C *4. Add 8 mL degassed HEGX containing (at least) 200 nmoles of mixed Tn5MEDS-A/-B oligonucleotides to the column (oligos, degassed? HEGX) *5. Leave column at room temp overnight *6. Wash with 20 volumes HEGX to remove free transposons at 4C. *7. Add 25 ml HEGX, 100 mM DTT to column. **a. 385.6 mg DTT in 25 mL (2.5 mL 1M DTT) **b. 12.5 mL 2X HEGX **c. 12.1 mL H20 **d. 25 mL Final volume *8. Run out 15 mL, stop flow and leave at 4C for at least 36 hours (over weekend) *9. Elute 6 fractions of 20 drops, then 12x30 drop fractions. *10. (optional) Leave the column in the remaining HEGX, 1M DTT for 24h. (Checked: Up to 30% may be in elute2).
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