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==April 2013== ===April 3, 2013=== * After meeting with Vipul again, we decided to try a library prep together. ===April 4, 2013=== * Vipul and I started a new celseq run with 3 RT reactions, each with 33 ng of brain ref RNA and 33 pmol of primer #39. IVT for 13 hrs. ==April 5, 2013== * Mg frag and TBU gel. IVT worked well. * Also ran a +/- control TBU gel for IVT (cDNA template with and without IVT reaction). * Phosphatase. * PNK. * Column purification using Zymo RNA Clean and Concentrator. [[File:2013_04_05 ivt positive negative control.jpg|400px]] ===April 6, 2013=== * 3' adapter ligation. * RT reaction. * 12 cycles of PCR, along with NTC and no adapter sample. * Nothing on TBE gel. Something went wrong, or something critical wasn't added. Phophotase and PNK are not suspects at this time because Rui and Brandon use those same enzymes without trouble. * T4 RNA Ligase 2, truncated may have lost activity, but our results were so cleanly negative that we doubt it. * I personally suspect one of the washes on the zymo column. I used 80% EtOH as per celseq protocol, but I think I should stick with the wash supplied by zymo, which I have used before successfully. ===April 9, 2013=== * Vipul brought fresh RNA samples for A100 (at 1 ng/ul, 100 pg/ul, and 50 pg/ul) and a new samples, SFM (at 1 ng/ul, 100 pg/ul, and 50 pg/ul). We will also use A3 (at 50 pg/ul) from 3/14 already in the freezer. * We want the following sample setup: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''Barcode''' |- | SFM_1ng||1||38 |- | A100_1ng_1||2||39 |- | A100_1ng_2||3||40 |- | SFM_100pg||4||38 |- | A100_100pg_1||5||39 |- | A100_100pg_2||6||40 |- | SFM_50pg||7||38 |- | A100_50pg_1||8||39 |- | A100_50pg_2||9||40 |- | A3_50pg_1||10||38 |- | A100_50pg_3||11||39 |- | A3_50pg_2||12||40 |- | |} ===April 11, 2013=== * No PCR products on agarose gel. Same as before. * Ran a TBU gel to see if there was any RNA going into RT after ligation. [[File:2013_04_11 check for rna after ligation.jpg|400px]] ===April 12, 2013=== * Start CEL-Seq for 100 ng and 0.5 ng libraries. IVT 14 hours overnight. ===April 13, 2013=== * Mg2+ frag and aRNA column. * BE day, poster judge. ===April 14, 2013=== * Finished CEL-Seq through PCR. Will run gel tomorrow. ===April 15, 2013=== * CEL-Seq is working again, for a 100 ng library and a 0.5 ng library. I used a new TruSeq kit and a different Zymo RNA Clean and Concentrator. I also took samples of the 100 ng library along the way. [[File: 2013_04_15 cel seq validation 1.jpg|400px]] [[File: 2013_04_15 cel seq validation 2.jpg|400px]] [[File: 2013_04_15 cel seq validation 3.jpg|400px]] * I gel purified both libraries so I can do qPCR for some housekeeping genes like GAPDH and beta actin. ===April 16, 2013=== * I am doing a second run with the new kit to make sure I can reproduce my results. I am also going to do bead purification instead of columns to see if I get better yield. I will take samples of the 100 ng library along the way, as before. ===April 21, 2013=== [[File: 2013_04_21 cel seq pcr.jpg|400px]] [[File: 2013_04_21 cel seq pcr cut.jpg|400px]] ===April 22, 2013=== [[File: 2013_04_22 cel seq gel results from 4_15 and 4_21.jpg|400px]] ===April 23, 2013=== [[File: 2013_04_23 gel frag.jpg|400px]] [[File: 2013_04_23 gel frag cut.jpg|400px]] ===April 24, 2013=== [[File: 2013_04_24 gel frag size selected.jpg|400px]] ===April 25, 2013=== [[File: 2013_04_24 lab meeting.pptx|April 25, 2013 progress report]]
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