Editing Tn5 Transposase (section)

== Protocol  ==

*Grow BL21(DE3)pLysS cells transformed with pWH1891 in 10 ml culture overnight<br> o Grow in 35 ug/ml chloramphenicol and 100 ug/ml carbenicillin <br> * Centrifuge and remove the supernatent to eliminate beta-lactamase from the medium.<br> * Inoculate 1 liter of LB/Cm/Amp culture medium with the culture and grow for 5-6 hours at 25 C until OD 0.5<br> * Induce cultures at OD 0.5 with 500 uM IPTG and grow an additional 3-5 hours<br> * Split and spin down cultures and resuspend in 80 ml cold water transferring to 50 ml centrifuge tubes<br> * Spin down a second time and resuspend in 2 x 5 ml cold TEGX + Roche Complete protease inhibitor<br> * Sonicate 3x pausing 10 minutes between sonications with the cells on ice<br> * Centrifuge at 4C to remove cell debris - 1 hour at 16,000 x g<br> * Load a 2 cm diameter column with 20 ml chitin bead suspension (10 ml beads)<br> * Wash the column with 100 ml cold TEGX buffer<br> * Load the cell supernatent onto the column and allow it to flow through<br> * Flow the supernatent past the column a second time<br> * Wash the column with 200 ml TEGX buffer<br> * Add 1 ml of 1M DTT to 20 ml of TEGX buffer<br> * Flow the 21 ml DTT + TEGX into the column<br> * Cap the column and hold at 4°C overnight<br> * Recover the protein with 10 ml TEGX buffer flowed through the column<br> * Recover a second 10 ml similarly<br> * Concentrate the protein by spinning in Centricon YM10<br> * Dilute concentrate with 40% glycerol to bring final to 50%<br> * Store aliquots at -80 and -20<br> * Dilute to final 100 ng/ul (1.8 pmol/ul) with storage buffer for use

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