Rui:LabNotes/SingleCell/2013-7-31: Difference between revisions

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===Results===
===Results===
[[File:8.1.13_IVT-TSO.jpg]]


===Libraries===
===Libraries===

Revision as of 01:38, 2 August 2013

mNPC plate with the latest version of hiMg-TSO and hiMg-IVT protocols

  • The latest version of hiMg-TSO and hiMg-IVT protocols confirmed on 7/29/13
  • I didn't use Betaine because it hasn't come in yet
  • mNPC plate was sorted on 7.10.13 in hiMg buffer [1]

Design

File:7.31.13 mNPC-display.jpg

Procedures

  • 2ul Lysis: the long procedure originally used for fluidigm
  • 3ul PNK: 1mM ATP, RNase-In (1:2d), PNK (1:2d)
  • 4ul PAP: 1mM ATP, PAP (1:10d)
  • 10ul RT: 3ul 0.2uM T20.id01-24 or RL.T7.id01-24
  • beads pools into 4 tubes, 3 column each (240ul beads + 240ul rec.)
  • TSO with TSO.r04 or TSO.r05 (LNA)
  • 2nd strand in 20ul
  • T7 addition for T20.id01-24 for 2 step IVT
  • beads purification
  • IVT in 20ul for 12 hrs
  • Directly take 2ul for a quick run (RT-PCR), purify the rest with Zymo kit
  • RT with N6 or N9
  • QPCR for 6-9 cycles
  • Redo RT-PCR with adjusted aRNA input
  • beads purification or size selection (if primer-dimer is an issue)

Results

File:8.1.13 IVT-TSO.jpg

Libraries