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=Agilent 15k Feb2017 Oligos= *[[Matt:LabNotes/2017-2-8|Probe design]] *Oligonucleotide Library received (Part Number G7221A) **Dan Konet says the pools yield ~10pmols *I resuspended to 200ul with nf-H2O (50nM concentration if actually 10pmols) ===Probe Expansion PCR=== *Expansion PCR in 4 tubes of 100ul **V4 primers for Human Brain and V7 primers for Mouse Brain {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''0 gap probes (ul)''' | align="center" style="background:#f0f0f0;"|'''20 gap probes (ul)''' |- | Agi15k (50nM)||10||10 |- | 2x Kapa SYBG qPCR MM||100||100 |- | 100uM pAP1V7U||0.8||0 |- | 100uM AP2V7||0.8||0 |- | 100uM pAP1V4U||0||0.8 |- | 100uM AP2V4||0||0.8 |- | H2O||88.4||88.4 |- | Total||200||200 |- | |} 94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 14 -> 72C 3min -> 15C hold *Stopped after 11 cycles **V7 could probably be stopped 2 cycles earlier but couldn't tell because qPCR software did weird "baseline subtraction" that messed with curve **Figured out to turn off baseline subtraction in settings for next time [[File:033017_ExpansionPCR_Agi15k.jpg| 550px]] *Purified with 2 Qiagen columns for each of the two probe sets *Eluted with 50ul each tube and combined into two tubes (100ul for each of the two probe sets) *Nanodrop: **V4: 42.2 ng/ul -> 42.2ng/ul / (195bp*660Da/bp) = 328nM **V7: 48.2 ng/ul -> 48.2ng/ul / (195bp*660Da/bp) = 375nM *Dilute with H2O to 25nM **V4: Add 1,188 H2O **V7: Add 1,372 H2O
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