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===Validation of results (round 3)=== *Successfully completely fragmented 6 ng or DNA *nanodrop did not give any values for A280. blanked with 2X Tn5 Dialysis buffer that was being used. Maybe something with higher TX100 concentrations allows nanodrop to measure protein concentrations better. '''8/7/2014 Transposase testing''' *with 6 ng Jurkat DNA (unconcentrated transposase) *already annealed sc1-T7tspn-NoP-bot to T7tspn-top2(10/30) 2. transposome complex generation, run controls!!! *add the below components into one tube and incubate for 20 minutes at RT 0.5 uL of annealed transposon 0.5 uL of 100% sterile glycerol 1.0 uL of Ez-TN5 transposase *store at -20, is good for a year 4. transposition reaction *add the below into one tube and incubate at 37C FOR 30 MINUTES. 1.0 uL washed cells 1.0 uL 2X Lysis Buffer 1.0 uL 5X custom tagmentation buffer 1.0 or 2.0 uL prepared transposomes 1.0 uL Nuclease free water ___________ 5.0 uL total solution 5. Protease digestion of transposase, protease inactivation To each tube, add: 1 uL Qiagen Protease, use 1 uL of 11.1 ug/uL (.5 AU/ml) protease (dilute 5.5 uL 20 ug/ul with 4.5 uL N-H2O) Incubate: 50C 10 minutes, 70C 20 minutes 6. Results *all fractions were working except the last one *will next test how much dilution can be do, don't know transposase concentration since nanodrop didn't work. can probably estimate concentration from dilution. [[File:ZhangLab 2 2014-08-07 19hr 59min-labeled.jpg|600px]] '''8/8/2014 REPEAT OF ABOVE PROTOCOL BUT WITH TRANSPOSASE DILUTIONS, AND ADDING SAME AMOUNT OF TRANSPOSON TO EACH DILUTED TRANSPOSASE SOLUTION (Round 3)''' *first fraction is more concentrated, don't know how much more concentrated though then the [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2014-7-2 transposase from the isolation done previously on 7/2/2014]. Thus will run the same dilution test on it. Also running IVT to see how yield compares to nextera with 8X less TX100, or 0% added during the column cleavage incubation. samples 1. f1, 0.025% TX100 - undil tsase, 6 ng DNA 2. f1, 0.025% TX100 - 2X tsase, 6 ng DNA 3. f1, 0.025% TX100 - 4X tsase, 6 ng DNA 4. f1, 0.025% TX100 - 8X tsase, 6 ng DNA 5. f2, 0.025% TX100 - undil tsase, 6 ng DNA 6. f2, 0.025% TX100 - 2X tsase, 6 ng DNA 7. f2, 0.025% TX100 - 4X tsase, 6 ng DNA 8. f2, 0.025% TX100 - 8X tsase, 6 ng DNA 9. 6 ng pure DNA 10. f1, 0.0% TX100 - undil tsase, 6 ng DNA 11. f1, 0.0% TX100 - 2X tsase, 6 ng DNA 12. f1, 0.0% TX100 - 4X tsase, 6 ng DNA 13. f1, 0.0% TX100 - 8X tsase, 6 ng DNA 14. 6 ng pure DNA [[File:ZhangLab 2 2014-08-08 16hr 38min-labeled.jpg|600px]] [[File:ZhangLab 2 2014-08-08 16hr 40min-labeled.jpg|600px]] *Example of Tn5 band from gosta [[File:ZhangLab 2 2014-06-11 Gosta SDS-PAGE gel image.png|600px]]
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