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==Test 50 cell level== * Test different isolation condition for 50 cells (sperm, ES) * NC: NTC; 150pg gDNA (~25 cells) * PC: 10pg, 100pg, 500pg RNA ===Samples=== * Zymo microprep kit for total RNA isolation on 1 tube of sperm and 1 tube of ES (elute with 7ul, 3.3ul each mixed with lysis buffer (-NP40) * 10ul lysis buffer (+ 0.45% NP40)is directly added into 1 tube of sperm and 1 tube of ES (carryover PBS is huge for sperm), mix and RT for 5min, then 4.55/5 ul each is continued with R.T. * 5 controls (0.5ul in 4.05ul lysis buffer with NP40) ===Procedure=== # Isolation of total RNA, elute with 7ul # 10ul lysis buffer at R.T. for 5 min # Lysis: 70C, 90sec # RT: 42C, 15min; 70C, 15min # ExoI-SAP: 37C, 30min, 85C, 15min # A-tail: 37C, 15min, 70C, 10min (TdT was not enough,so I added 3.2 instead of 4.2ul. But considering the glycerol left in the enzyme tube, I am really uncertain how much I added.) # 5 cycles of PCR: 95C, 3min; 50C, 2min; 72C, 3min (X 2) | 95C, 30sec; 50C, 1min; 72C, 2min (X 3) # 25-30 cycles of PCR: 95C, 3min| 95C, 30sec; 67C, 1min; 72C, 2min+6sec/cycle (X 25-30)| 15C hold ===QPCR curve=== Ctrols: 500pg RNA and 150pg gDNA are stopped at 19 cycles, 10pg and 100pg RNA are stopped at 29 cycles [[File:Ctrols_5.19.11.jpg]] Samples: Samples in the direct lysis method are stopped at 19 cycles, RNA samples are stopped at 29 cycles [[File:Samples_5.19.11.jpg]] ===TBE-PAGE=== After 20 cycles: NTC, 500pg RNA, 150pg gDNA (25cells), sperm, sperm, ES, ES (50 sperms or ES cells are in lysis buffer directly and split into 2 tubes for processing.) [[File:PCR.1_5.19.11.jpg]] After 30 cycles: NTC, 10pg RNA, 100pg RNA, sperm RNAs (2), ES RNAs (2) [[File:PCR.2_5.19.11.jpg]]
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