Rui:LabNotes/SingleCell/2011-5-19: Difference between revisions

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===TBE-PAGE===
===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]]

Revision as of 00:54, 20 May 2011

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

  1. Isolation of total RNA, elute with 7ul
  2. 10ul lysis buffer at R.T. for 5 min
  3. Lysis: 70C, 90sec
  4. RT: 42C, 15min; 70C, 15min
  5. ExoI-SAP: 37C, 30min, 85C, 15min
  6. 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.)
  7. 5 cycles of PCR: 95C, 3min; 50C, 2min; 72C, 3min (X 2) | 95C, 30sec; 50C, 1min; 72C, 2min (X 3)
  8. 25-30 cycles of PCR: 95C, 3min| 95C, 30sec; 67C, 1min; 72C, 2min+6sec/cycle (X 25-30)| 15C hold

QPCR curve

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