Matt:LabNotes/2015-1-12

From ZhangLabWiki
Jump to navigation Jump to search

Make First Strand cDNA for in vitro Capture with CA12k_Nov2014 Probe Set[edit]

  • Followed NEB E6300S protocol: [1]
  • 2 tubes for RNA sample and 1 tube with no RNA (replace with H2O) as negative control
Components Volume
RNA 1 ul (0.5 ug)
d(T)23VN (50 µM) 2 ul
H2O 5 ul
Total 8 ul
  • Denatured RNA for 5 min at 70C and then put on ice
  • Added the following to tubes
Components Volume
M-MuLV Reaction Mix 10 ul
M-MuLV Enzyme Mix 2 ul
Total 20 ul
  • Incubated for one hour at 42C
  • Inactivated enzyme for 5 min at 80C
  • Purified cDNA in one Qiagen PCR clean up column and NTC in another
    • Eluted 30ul each and stored in -20C