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==Sanger validation of candidate mutations== ===DNA preparation=== * prepared the following stock solutions for PCR templates Sample Conc. Volume H2O Final Conc. CV-iPS F 309ng/ul 1ul 153.5ul 2ng/ul CV-ips B 358ng/ul 1ul 178ul 2ng/ul CV-Fibr 712ng/ul 1ul 355ul 2ng/ul ===PCR=== *Primers and templates: {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''CV-fibroblast''' | align="center" style="background:#f0f0f0;"|'''CV-ips-B''' | align="center" style="background:#f0f0f0;"|'''CV-ips-F''' |- | MCM3||x||x||x |- | 18-22778270||||||x |- | ZSCAN||x||x||x |- | C1orf||x||x||x |- | SPEN||x||x||x |- | |} *13 reactions total. DNA(2ng/ul) 3ul Taq 2x master mix 25ul 10uM Primer mix 1ul H2O 21ul total 50ul 94C 2min -> 35x (94C 30s -> 57C 30s -> 72C 30s) -> 72C 3min. *use 3ul to run a TBE gel to check the PCR results, then save 22ul for backup, and use the rest to do Qiaquick column purification elute in 30ul EB. *Use Nanodrop to check the concentration of the purified PCR product. *TBE gel result: [[Image:ZhangLab_2 2009-12-04 10hr 21min.jpg]] [[Image:ZhangLab_2 2009-12-03 15hr 14min.jpg]] *Prepare the samples for sequencing: *USE Pre-Mixed Samples Preparation Guidelines from Genewiz to mix the DNA and custom primers. *The DNA size is 200-500bp, so we use 3-10ng of DNA in 8ul volume {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''CV-fibroblast (DNA+H2O)''' | align="center" style="background:#f0f0f0;"|'''CV-ips-B (DNA+H2O)''' | align="center" style="background:#f0f0f0;"|'''CV-ips-F (DNA+H2O)''' |- | MCM3||1ul + 7ul||2ul + 6ul||1ul + 7ul |- | 18-22778270||||||1ul + 7ul |- | ZSCAN||1.2ul + 6.8ul||1.5ul + 6.5ul||1.5ul + 6.5ul |- | C1orf||1ul + 7ul||1.1ul + 6.9ul||1.2ul + 6.8ul |- | SPEN||2ul + 6ul||1.1ul + 6.9ul||2ul + 6ul |- | |} *Add the Forward primer for sequencing, dilute the Forward primer to 2uM, add 4ul of primer and get a final volume of 12ul. *[[Media:|Raw ABI trace file]].
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