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===Results=== *total RNA. {| {{table}} | align="center" style="background:#f0f0f0;"|'''9/25/2015''' | align="center" style="background:#f0f0f0;"|'''intensity''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||1540.165708||5|||||||||| |- | ||441.3047926||2.5||in 10 ul||for 50 ng|||||| |- | 1. i502_T7-1_long-i701_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep1||3737.306598||9.998678313||99.98678313||5.000660931|||||| |- | 2. i502_T7-1_long-i702_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep2||3910.542279||10.3928039||103.928039||4.811021211|||||| |- | 3. i502_T7-1_long-i703_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep3||10229.91571||24.76990419||247.6990419||2.01857866|||||| |- | 4. i503_T7-1_long-i701_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep4||4889.326632||12.61961973||126.1961973||3.962084521|||||| |- | 5. i503_T7-1_long-i702_long, T7-nXTv2_i5-1, 5X tn5059, 6 ng pure DNA||97614.73967||223.5776485||2235.776485||0.223635951||/4||55.89441214||0.894543803 |- | 6. i503_T7-1_long-i703_long, T7-nXTv2_i5-1, 5X tn5059, NTC||257.4794671||2.081782092||20.81782092||1 uL|||||| |- | ||0||1.495994883||14.95994883||0|||||| |- | ||-83.77946196||1.305389637||13.05389637||38.30274009|||||| |- | ||||||||#DIV/0!|||||| |- | |} *TBU gel after IVT, Guanidine HCl to remove proteins *looks like it worked fine with 333 cells [[File:ZhangLab 2 2015-09-25 13hr 32min-labeled.jpg|600px]] *qPCR curves *curves didn't go up, something wrong? (was to much DMF in PCR reaction) [[File:2015-09-25 THS minor protocl changes validation.png|600px]] *TBE after PCR amplification *can tell that fragmentation as occured, after comparing to gel run of what is left over on beads [[File:ZhangLab 2 2015-09-25 20hr 33min-labeled.jpg|600px]] *running of left over beads after elution on TBE *can tell DNA is still intact at this point [[File:ZhangLab 2 2015-09-25 20hr 13min-labeled.jpg|600px]] *'''9/28/2015 UPDATE''' *cleaned with zymo clean and concentrator and PCR worked very well. DNA fragments were present in solution, just DMF did not allow PCR to run correctly. after cleaning and readdin primers, worked fine. *also used KAPA SYBR fast, and 50 uL reaction volume *will try different methods of RNA processing to see which gives best smears [[File:ZhangLab 2 2015-09-28 15hr 11min-labeled.jpg|600px]] *gel size selection amounts *gel size selection *gel size selection validation
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