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===IV. Chitin column=== '''Without transposons''' *1. Prepare chiten columns. pour 12 mL of chitin resin into column, after draining put upper bed support on top, and wash with 5 column volumes of buffer. Now ready for use **a. OR take 12 mL of resin, let settle or spin down in 50 mL conical at 200xg for <1 minute., remove supernatant, and wash with 4 volumes column buffer. Repeat wash 5X. **b. Add supernatant from PEI to washed chitin resin in conical. Lightly mix and let sit for 3.5 hours. **c. Add solution from conical to chromatography column, after draining put upper bed support on top, and wet with buffer. **d. proceed to step 3. *2. Add supernatant from PEI precipitation to 10ml Chitin column at 0.4ml/min in HEGX (~3.5 hours) (Chitin Resin, column?,lots of HEGX) '''Incubated for 1 hr 45 mins''' *3. Wash with 20-30 volumes (200-300 ml) HEGX at gravity flow (2-4 ml/min) OR wash overnight at 0.2ml/min *4. Add 20 ml HEGX, 100 mM DTT to column. ** . 308 mg DTT in 22 mL **a. 10 mL 2X HEGX **b. 2.0 mL 1M DTT '''added 309 mg DTT to solution''' **c. 8.0 mL H20 **d. 20 mL Final volume *5. Run out 5 mL, stop flow and leave at 4C for at least 36 hours (over weekend) ''' did for 67 hours, 30 mins''' *6. Elute 4 fractions of 20 drops, then 12x40 drop fractions. Test concentration (nanodrop) *7. Leave the column in the remaining HEGX, 1M 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) (Chitin Resin, column?,lots of HEGX) *2. Wash with 10-20 volumes HEGX, (100-200 mL) at gravity flow (~4 mL a minute = 25 mins) *3. Repeat wash with 2 column volumes (200 mL) degassed HEGX at gravity flow *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 *7. Add 20 ml HEGX, 100 mM DTT to column. **a. 308 mg DTT in 22 mL **b. 10 mL 2X HEGX **c. 2.0 mL 1M DTT **d. 8.0 mL H20 **e. 20 mL Final volume *8. Run out 15 mL, stop flow and leave at 4C for at least 36 hours (over weekend) *9. Elute 4 fractions of 20 drops, then 12x40 drop fractions. Test concentration (nanodrop) *10. Leave the column in the remaining HEGX, 1M DTT for 24h. (Checked: Up to 30% may be in elute2).
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