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==Library construct by ligation with Y adaptor== ===Procedure=== # Digestion with BciVI in 30ul or 50ul (SL) # Beads purification 1X # Ligation with Y adaptor (1:10):1.1ul, 0.7ul, 0.5ul, 0.7ul # Beads purification (up to 50ul, 0.7X) # Phusion PCR with N2.R.ID5 - ID8 ===Concentration=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''>Digestion''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''260/280''' | align="center" style="background:#f0f0f0;"|'''260/230''' |- | sperm RNA||39.12||1.9||2.77 |- | ES RNA||51.57||1.92||1.92 |- | sperm lysis||16.27||1.73||1.42 |- | ES lysis||45.64||1.93||1.99 |- | |||||| |- | |||||| |- | >Ligation||ng/ul ||260/280 ||260/230 |- | sperm RNA||35.03||2.05||2.19 |- | ES RNA||45.66||1.92||1.99 |- | sperm lysis||13.99||2.04||1.7 |- | ES lysis||39.23||2.06||2.12 |- | |||||| |- | |||||| |- | >PCR||ng/ul ||260/280 ||260/230 |- | sperm RNA ID5||21.29||1.73||1.69 |- | ES RNA ID6||21.31||1.72||1.7 |- | sperm lysis ID7||19.24||1.61||1.14 |- | ES lysis ID8||20.03||1.74||1.1 |- | |} ===QPCR curve=== Original 10 cycles: [[File:Phusion 10 cycles_5.23.11.jpg]] Additional 5 cycles: [[File:Phusion 5 cycles_5.23.11.jpg]] ===TBE PAGE=== [[File:5.23.11_Phusion PCR.jpg]] PCR failed, highly doubt if Y-adaptor is too low (invisible in the gel?)
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