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===Validation of results=== *Successfully completely fragmented 6 ng of DNA. *f1 1.5606 average abs; 18.04 uM, 2.2 mL total, final of 0.9 mL, 2.44X concentration, '''25.92 uM''' *f2 1.5355 average abs; 17.75 uM, 2.2 mL total, final of 0.9 mL, 2.44X concentration, '''25.02 uM''' *calculation for uM. 100 uM/8.65 = X uM/abs '''9/9/2014''' *with 6 ng DNA *annealed sc1-T7tspn-bot(8/22 NoP) to T7tspn-top2(8/22) *tsp complex made by incubating at RT for ~30 mins *transposition reaction at 37C for 30 mins, followed by qiagen protease digestion, 50C for 10 mins, 70C for 20 mins. *reaction composition 1 uL 5X custom tagmentation buffer (buffer D6) 1 uL pure Jurkat DNA (6 ng) 1 uL undiluted or diluted transposome complex 1 uL 2X Lysis buffer 1 uL H2O __________ 5 uL total samples. washed with .5% TX100, 1 M NaCl. eluted .05% TX100, 0.8 M NaCl 1. f1, undil tsase, 6 ng DNA 2. f1, 2X dil. tsase, 6 ng DNA 3. f1, 4X dil. tsase, 6 ng DNA 4. f1, 8X dil. tsase, 6 ng DNA 5. f2, undil tsase, 6 ng DNA 6. f2, 2X dil. tsase, 6 ng DNA 7. f2, 4X dil. tsase, 6 ng DNA 8. f2, 8X dil. tsase, 6 ng DNA 9. 6 ng pure DNA [[File:ZhangLab 2 2014-09-09 12hr 42min-labeled.jpg|600px]] [[File:ZhangLab 2 2014-09-09 12hr 43min-invert-labeled.jpg|600px]] '''9/10/2014 transposase accidentally left overnight (~16 hours) at RT.''' *same assay for activity that was done above *appears to be very small loss of activity, if any when compared to assay above. Freshly ordered jurkat DNA was used. 4X dilution of transposase still completely fragments DNA. samples 1. f1, undil tsase, 6 ng DNA 2. f1, 2X dil. tsase, 6 ng DNA 3. f1, 4X dil. tsase, 6 ng DNA 4. f1, 8X dil. tsase, 6 ng DNA 5. f1, 16X dil. tsase, 6 ng DNA 6. f2, undil tsase, 6 ng DNA 7. f2, 2X dil. tsase, 6 ng DNA 8. f2, 4X dil. tsase, 6 ng DNA 9. f2, 8X dil. tsase, 6 ng DNA 10. f2, 16X dil. tsase, 6 ng DNA 11. 6 ng pure DNA [[File:ZhangLab 2 2014-09-10 15hr 48min-labeled.jpg|600px]] [[File:ZhangLab 2 2014-09-10 15hr 49min-invert-labeled.jpg|600px]] '''9/9/2014 IVT testing of transposase'''
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