Matthew Cai/LabNotes/2013-3-8

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First Strand cDNA synthesis[edit]

  • Followed NEB E6300S protocol: [1]

Got 5ul aliquot of Human Brain Reference RNA from Rui at concentration 1ug/ul

  • Used 1 ul for cDNA synthesis and put the rest in my box in -20C (Top of tube labelled HBBR)
  • Mixed components in 0.2 ml microfuge tube labelled HBBR 1ug cDNA
Components Volume
RNA 1 ul (1 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
    • Tightened the thermocycler too much and bent the tube slightly out of shape
  • Added the following to tube
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
  • Stored at -20C for Second Strand synthesis on Saturday 3/9/13