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==Agilent Probe Prep== *Dr. Zhang resuspended probes to 20nM *Dr. Zhang aliquoted Agilent probes into separate tube labelled "FISHYB V1 probes 5/07/13" ===Probe Expansion PCR=== Expansion PCR in 2 tubes of 180ul total (1 tube for 0bp gap probes with AP1V6 primers; 1 tube for 20bp gap probes with AP1V4 primers) <br> {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''0 gap probes (ul)''' | align="center" style="background:#f0f0f0;"|'''20 gap probes (ul)''' |- | Agi26k (20nM)||10||10 |- | 2x Kapa SYBG qPCR MM||100||100 |- | 100uM pAP1V6U||0.8||0 |- | 100uM AP2V6||0.8||0 |- | 100uM pAP1V4U||0||0.8 |- | 100uM AP2V4||0||0.8 |- | H2O||88.4||88.4 |- | Total||200||200 |- | |} *NOTE: Mixed thoroughly and then removed 20ul so there would be room to close lid 94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 14 -> 72C 3min -> 15C hold *Stopped after 12 cycles [[File:051013_ExpansionPCR_Agi26k.jpg| 650px]] *Purified with 2 Qiagen columns for each of the two probe sets *Eluted with 40ul each tube and combined into two tubes (80ul for each of the two probe sets) *Nanodrop: **0gap: 16.90 ng/ul -> 16.9ng/ul / (228bp*660Da/bp) = 112nM **20gap: 18.46 ng/ul -> 18.46ng/ul / (228bp*660Da/bp) = 123nM *Final Volume of 48.5ul for each first round amplicon Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-11
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