Reagent/Supplies:

Protocol:

DNA digestion:

  1. Keep extracted RNA on ice or thaw total RNA on ice (10 pg- 5 µg required total RNA).

  2. Thaw DNA-free reagents on ice.

  3. Add 0.1v 10 x DNase buffer and 1 µl DNase I to appropriate amount of total RNA and mix by flicking.

  4. Incubate 20 minutes at 37°C.

  5. Mix DNase Inactivation reagent well. If reagent is difficult to mix, add 1v H2O/0.1 mM EDTA.

  6. Add 0.1v DNase Inactivation reagent.

  7. Incubate 2 minutes, flicking intermittently.

  8. Spin 1 minute at 10,000 x g to pellet DNase Inactivation reagent.

  9. Remove supernatant and use, or store at -70°C.

cDNA synthesis:

  1. Keep on ice: oligo DT (20 mM), dNTP (20 mM), 5x SST buffer, DTT, SuperScript III, RNase Out, and DNA digested RNA.

  2. To 4 µl DNA digested RNA, add 8 µl of the following mastermix for 1 reaction(s) (enter # of reaction):

    • 1 µl oligo DT (20 µM)
    • 0.5 µl dNTP (20 mM)
    • 6.5 µl ddH2O
  3. Incubate 5 minutes at 65°C.

  4. Cool on ice.

  5. While cooling, make the following mastermix for 1 reaction(s) (enter # of reaction):

    • 4 µl 5x 1st strand buffer
    • 2 µl 100 mM DTT
    • 1 µl RNase OUT (RNase inhibitor)
  6. Add 7 µl of mastermix.

  7. Add 1 µl of SSTIII (SuperScript III). Flick and collect.

  8. Run sample in thermocycler:

    1. 42°C for 50 minutes

    2. 70°C for 15 minutes

    3. 4°C soak until needed

  1. Add 1 µl RNase H to remove complimentary strand.

  2. Incubate 20 minutes at 37°C.

  3. Estimate cDNA yield using a spectrophotometer.

  4. Dilute to appropriate concentration: 5 µl TaqMan template input.

TaqMan:

  1. Aliquot 5 µl sample and/or DNA template to optical tubes. Seal with Parafilm.

  2. Prepare Mastermix in PCR setup room. Click to download Mastermix Wizard

  3. Add 15 µl Mastermix to optical tubes and cap. Keep on ice and transfer to ABI 7700.

  4. Flick and spin to collect. Run on ABI 7700.

Department of Microbiology
School of Medicine
University of Washington
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