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===Validation of results=== *Successfully completely fragmented 6 ng or DNA, and partially fragmented 12 or 25 ng DNA. '''6/30/2014''' *'''Most concentrated fractions successfully completely fragmented 6 ng of pure DNA. second fraction had high fragmentation too.''' *Tn5 is unconcentrated and is currently in 2X dialysis buffer. used 6 or 12 ng pure Jurkat DNA. *Nextera positive control used 2 uL of 2.38X diluted nextera tn5 (6/18/14 sa nextera t7-top2) *annealed sc1-T7tspn-NoP-bot to T7tspn-top2(10/30) Protocol: 1. already annealed T7-top2 2. ~20 mins at RT to generate transposome complex 3. make master mix, tagmentation at 55C for 5 mins 4. Add 1 uL proteinase K and incubate for 10 mins at 50C 5. run on TBE gel reaction composition: 1 uL 5X custom tagmentation buffer (D6) 1 uL pure Jurkat DNA (6-12 ng) 1 uL tspsome 1 uL 2X Lysis Buffer 1 uL Nuclease free water _____________ 5 uL total [[File:ZhangLab 2 2014-06-30 19hr 58min-labeled.jpg|600px]] '''7/1/2014 fraction 2 dialyzed/concentrated into storage buffer''' *Second combined fractions after dialysis in storage buffer. used 6, 12, of 24 ng pure Jurkat DNA. *Nextera positive control was normal *lanes 3,6,9 have overflow into adjacent lanes because there was to much solution *annealed sc1-T7tspn-NoP-bot to T7tspn-top2(10/30) Protocol: 1. already annealed T7-top2 2. ~20 mins at RT to generate transposome complex 3. make master mix, tagmentation at 55C for 6 mins 4. Add 1 uL proteinase K and incubate for 10 mins at 50C 5. run on TBE gel reaction composition: 5uL 10uL 20uL 1 2 4 uL 5X custom tagmentation buffer (D6) 1 2 4 uL pure Jurkat DNA (6-24 ng) 1 1 1 uL tspsome 1 2 7 uL 2X Lysis Buffer 1 3 7 uL Nuclease free water __________________________ 5 10 10 uL total [[File:ZhangLab 2 2014-07-01 19hr 15min-labeled.jpg|600px]] *'''7/2/2014 fraction 1 dialyzed/concentrated into storage buffer''' *'''Fragmentation wasn't complete, while nextera was. earlier assay before concentrating showed complete fragmentation. could be two things. **'''1. took sample from top after putting into -20C and did not mix, thus could have gotten less protein''' **'''2. 37C for ~30 mins for making transposome complex (however worked for nextera)''' **'''3. Maybe concentrating caused some loss in activity, however opposite was shown with sample 2 on 7/1/2014.''' *'''will try again incubating at RT for complex formation, and mixing solution well before pipetting enzyme''' *after concentrating by dialysis: smpl orig A280 conc. before d after d dilution new conc 1 0.93 10.75 uM 2.2 mL 1.25 mL 1.8X 19.25 uM 2 0.83 9.59 uM 1.8 mL 1.0 mL 1.8X 17.26 uM *Tn5 is in Tn5 storage buffer *Nextera positive control is normal *annealed sc1-T7tspn-NoP-bot to T7tspn-top2(10/30) Protocol: 1. already annealed T7-top2 2. ~30 mins at 37C to generate transposome complex 3. make master mix, tagmentation at 55C for 6 mins 4. Add 1 uL proteinase K and incubate for 10 mins at 50C 5. run on TBE gel reaction composition: 1 uL 5X custom tagmentation buffer (D6) 1 uL pure Jurkat DNA (6-12 ng) 1 uL tspsome 1 uL 2X Lysis Buffer 1 uL Nuclease free water _____________ 5 uL total [[File:ZhangLab 2 2014-07-02 19hr 22min-labeled.jpg|600px]] *'''7/7/2014 repeat fragmentation test with mixing transposome solution and incubation at RT for ~40 minutes''' **'''Worked correctly, thus not mixing the tn5 solution was probably the issue.''' **'''Next time purify DNA before running on gel''' **'''Second fraction has slightly less activity then the first, however this can be fixed by adding more transposome complex to the reactions''' *annealed sc1-T7tspn-NoP-bot to T7tspn-top2(10/30) Protocol: 1. already annealed T7-top2 2. ~40 mins at RT to generate transposome complex 3. make master mix, tagmentation at 55C for 6 mins 4. Add 1 uL proteinase K and incubate for 10 mins at 50C 5. run on TBE gel reaction composition: 1 uL 5X custom tagmentation buffer (D6) 1 uL pure Jurkat DNA (6-12 ng) 1 uL tspsome 1 uL 2X Lysis Buffer 1 uL Nuclease free water _____________ 5 uL total [[File:ZhangLab 2 2014-07-07 14hr 06min-labeled.jpg|600px]] [[File:ZhangLab 2 2014-07-07 14hr 09min-labeled.jpg|600px]] '''8/13/2014 update, tn5 tspase dilution''' *diluted transposase to see how much it fragmented DNA. *f1 and f2 for round 2 are just as good as for round 3, but less contaminatined. [[File:ZhangLab 2 2014-08-13 19hr 50min-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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