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Arichard:Notebook/2013/February
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==February== ===February 7=== * Diluted the first 32 of the 48 200 uM celseq RT primers to 3 different concentrations corresponding to 3 different RNA loadings: {| {{table}} | align="center" style="background:#f0f0f0;"|'''RNA''' | align="center" style="background:#f0f0f0;"|'''1 ng''' | align="center" style="background:#f0f0f0;"|'''100 pg''' | align="center" style="background:#f0f0f0;"|'''10 pg''' |- | [RT primer]||32 pmol/ul||3.2 pmol/ul||0.32 pmol/ul |- | |} * See reagents for exact layout ===February 8=== * Processed first 16 samples from total RNA. Performed first strand RT and second strand synthesis. Pooled samples for EtOH precipitation and left in -80 C until I get the other 16 samples ready. * Sample layout is as follows: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''RNA input''' | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''RNA input''' | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''RNA input''' | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''RNA input''' |- | 1||1 ng ||9||100 pg||17||100 pg||25||10 pg |- | 2||1 ng ||10||100 pg||18||100 pg||26||10 pg |- | 3||100 pg||11||100 pg||19||10 pg||27||10 pg |- | 4||100 pg||12||100 pg||20||10 pg||28||10 pg |- | 5||100 pg||13||100 pg||21||10 pg||29||10 pg |- | 6||100 pg||14||100 pg||22||10 pg||30||10 pg |- | 7||100 pg||15||NTC||23||10 pg||31||NTC |- | 8||100 pg||16||NTC||24||10 pg||32||NTC |- | |} ===February 14=== * Processing second batch of 16 samples through second strand synthesis. I will end up with 2 EtOH precipitation reactions, each with 16 samples. * Do CS primer reaction and IVT tomorrow. ===February 21=== * IVT reaction and Mg2+ fragmentation * Richard Szubin ran a bioanalyzer on for my samples (Andrew 7: #1-16, Andrew 14: #17-32). [[File: bioanalyzer_2_21_2013.pdf|400px]] ===February 28=== * Starting a new run with Vipul Bhargava's samlples: A3 (50 pg/ul) and A100 (50 pg/ul). * 9 samples total: 1 pg of A3-1, A3-2, and A100; 10 pg of A3-1, A3-2, and A100; 50 pg of A3-1, A3-2, and A100. {| {{table}} | align="center" style="background:#f0f0f0;"|'''Vipul's samples''' | align="center" style="background:#f0f0f0;"|'''''' |- | 1||A3-1, 1 pg |- | 2||A3-2, 1 pg |- | 3||A100, 1 pg |- | 4||A3-1, 10 pg |- | 5||A3-2, 10 pg |- | 6||A100, 10 pg |- | 7||A3-1, 50 pg |- | 8||A3-2, 50 pg |- | 9||A100, 50 pg |- | |} * I will also have 16 samples of total RNA as follows: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''RNA (pg)''' | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''RNA (pg)''' |- | 1||100||9||10 |- | 2||100||10||10 |- | 3||10||11||10 |- | 4||10||12||10 |- | 5||10||13||10 |- | 6||10||14||10 |- | 7||10||15||NTC |- | 8||10||16||NTC |- | |} * I will also run an NTC for IVT with a 5 ul rxn vol, as well as a 5 ul positive control rxn using 2 ul of template from Brandon. * In total, I will have 4 rxns going in to IVT: AR 2/28, VB 2/28, +, and -. * Instead of using Phusion Flash to add the T7 promoter and 5' AD, I am switching to Bst Large Fragment (no 3' to 5' exonuclease activity). After EtOH precipitation, I will resuspend the ds cDNA in 4.76 ul of H2O. I will then add 0.64 ul 10X NEB Buffer 2 and 0.5 ul of 10 pmol/ul T7 primer. The Tm for 50% is 69 degC and the Tm for 90% is 66 degC. I will denature for 2 min @ 95 degC, followed by annealing for 10 min @ 66 degC. I will then add 0.5 ul Bst and incubate for 30 min @ 65 degC, followed by 20 min @ 80 degC to heat inactivate.
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