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===Results=== *Had leftover RNA generated from [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2013-5-10 transposition on pure Jurkat DNA]. Used sample #2 after IVT but before purification, has been frozen at -20C. Purified with AMPURE RNA beads, adding 30 uL to solution and 1:1.8X ratio of sample:beads. *flow though has most of the transposon fragment which is good. *quantification of RNA from gel {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''intensity''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||5||2504.210075|||| |- | ||2.5||1036.780031||in 10 uL||for 200 ng |- | 2013-5-10 pure Jurkat DNA elution||158.6959685||92719.44276||1586.959685||1.260271461 |- | 2013-5-10 pure Jurkat DNA on beads||112.9738386||65881.83196||1129.738386|| |- | |} [[File:ZhangLab 2 2013-07-10 14hr 59min-labeled.jpg|600px]] *TBE gel after second strand synthesis and AMPURE DNA beads purification *look as expected, similar to TBU gel on IVT RNA. *eluted samples in 20 uL N-H2O, concentrated to 10 uL, ran gel on 1 uL of the sample then. *qunaitation of samples: {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''intensity''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||5||5401.670161|| |- | ||2.5||3596.240107||in 10 uL |- | Index 49, NXTA adaptor, 1. DNAPI||60.64528949||45587.14136||606.4528949 |- | Index 50, NXTA adaptor, 2. Klenow exo-||77.69815224||57902.24173||776.9815224 |- | Index 51, NXTA adaptor, 3. BST 2.0||75.27544685||56152.63167||752.7544685 |- | Index 52, NXTA adaptor, 4. BST 2.0 no buf||77.8642761||58022.21173||778.642761 |- | Index 53, NXTA adaptor, 5. taq2x||50.21377179||38053.79113||502.1377179 |- | Index 54, NXTA adaptor, 6. NTC||||||0 |- | |} [[File:ZhangLab 2 2013-07-12 18hr 54min-labeled.jpg|600px]] *qPCR from barcode addition. curves didn't go up, but added in SYBR green manually and might have done that incorrectly. Also didn't clean reaction since the transposition reaction so that may have contributed to SYBR green not working correctly. Gel after barcode addition looks good. *samples in order Index 49, NXTA adaptor, 1. DNAPI Index 50, NXTA adaptor, 2. Klenow exo- Index 51, NXTA adaptor, 3. BST 2.0 Index 52, NXTA adaptor, 4. BST 2.0 no buf Index 53, NXTA adaptor, 5. taq2x Index 54, NXTA adaptor, 6. NTC [[File:2013-07-12 No Rnase III no PAP protocol testing.bmp|600px]] *after custom nextera tagmentation and barcode addition *when compared to gel after second strand synthesis, can definetly see high molecular weight fragments have been reduced, and there are more fragments between ~200-400 bp. [[File:ZhangLab 2 2013-07-15 11hr 13min-labeled.jpg|600px]] *amounts used for gel size selection {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''intensity''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | 7/15/2013||5||6162.250184|||| |- | ||2.5||2898.130086||||for 100 ng |- | Index 49, NXTA adaptor, 1. DNAPI||9.098669779||11513.67034||||10.99061758 |- | Index 50, NXTA adaptor, 2. Klenow exo-||13.92165423||17810.79053||||7.183054424 |- | Index 51, NXTA adaptor, 3. BST 2.0||13.49057326||17247.95051||||7.412583442 |- | Index 52, NXTA adaptor, 4. BST 2.0 no buf||13.92723766||17818.08053||||7.180174735 |- | Index 53, NXTA adaptor, 5. taq2x||10.32603734||13116.18039||||9.68425706 |- | Index 54, NXTA adaptor, 6. NTC|||||||| |- | |||||||| |- | ||||100|||| |- | ||ng total||500|||| |- | ||sum||42.45068724|||| |- | ||dye to add||8.254300296|||| |- | ||per well||12.67624688|||| |- | |} *gel size cut validation [[File:ZhangLab 2 2013-07-15 12hr 59min.jpg|600px]] *gel size selection validation of size range [[File:ZhangLab 2 2013-07-15 16hr 33min.jpg|600px]]
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