Matt:LabNotes/2013-3-11

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Needed to synthesize more cDNA from 1ug total RNA to run 2 sets of probe capture reactions in parallel due to 2 types of primers and not knowing which will work better

First Strand cDNA synthesis[edit]

  • Followed NEB E6300S protocol: [1]

From the 4ul of Human Brain Reference RNA 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

Second Strand cDNA synthesis[edit]

  • Follow NEB E6111S protocol: [2]
  • Added 48 ul H20
  • Added 8ul 10x Second strand synthesis reaction buffer
  • Added 4ul Second strand synthesis enzyme mix
  • Mixed by pipetting
  • Incubated for 2.5 hours at 16C
  • Purified using Qiagen minelute column (eluted 23ul into 1.5ml tube labelled HBRR cDNA 3.11.13)
  • Measured DNA conc with Nandrop: 36.1 ng/ul
    • 21.5ul (0.776 ug) sample left in tube

cDNA + RNase digestion[edit]

  • Did 2 more first strand and second strand cDNA reactions like above
    • Stopped before purification to digest RNA and then purify tomorrow
    • Stored samples in -20C freezer