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==Additional 10 cycles of direct lysis samples== * Continue with lab note on 5.19.11 * Additional 10 cycles for direct lysis samples (sperm and ES) in parallel with 500pg RNA and 150pg gDNA ===QPCR curve=== [[File:additional 10 cycles_5.20.11.jpg]] ===TBE PAGE=== [[File:10 cycles_5.20.11.jpg]] ==Repeat 2nd strands and PCR with the remaining samples== * Continue with samples on 5.19.11, the remaining 9ul A-tailing product * 2nd strand with P8 * PCR with 30 cycles ===QPCR=== [[File:5.20.11_remaining samples.jpg]] ===Beads purification=== * Combined into 4 samples, 8 PCR tubes per samples * Sperm RNA, ES RNA, Sperm lysis, ES lysis * 0.7X beads purification * Elute with 50ul water {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''260/280''' | align="center" style="background:#f0f0f0;"|'''260/230''' |- | sperm RNA||43.64||1.9||1.68 |- | ES RNA||295.67||1.93||2.18 |- | ES lysis||220.08||1.92||1.97 |- | sperm lysis||23.5||1.68||0.95 |- | |} ===Cloning=== * Ligation: ~25-30ng purified PCR + 2.5ng vector in 10ul total ligation, 14C, o/n * Transformation, using Kan30 plate
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