Applied Biosystems quantitative PCR: StepOne Software v2.1

Computer login: Administrator

Password: realtime

  1. Open StepOne v2.1 software (icon on Desktop). Username is ‘Guest’ (no password)
  2. Click on “Advanced Setup”; in “experimental properties”:

·  Name your experiment

·  Select “StepOnePlus Instrument (96 wells)”

·  Select “Quantitation – Comparative CT (DDCT)

·  Select “SYBR green reagents” and check box to include melt curve (þ)

·  Select “Standard ~ 2 hrs to complete run”

  1. Go into “Plate Setup”

·  Define your targets (i.e. m18S and Agg)

·  Define your samples (i.e. 21% +RT, 21% -RT, and –RNA+RT)

·  Click on tab that says “assign targets and samples” – label your experimental plate appropriately with targets/types you have designated

  1. Go into “Run Method”

·  Type in reaction volume (i.e. 20 ul)

·  Set up your program. Can set up and save programs.

·  Program we use (Richa’s Protocol - 60):

Ø  Holding stage

  1. 95°C for 15 min

Ø  Cycling stage (40 cycles)

  1. 94°C for 15 sec
  2. 60°C for 20 sec (plate read here)
  3. 72°C for 20 sec

Ø  Melt Curve stage

  1. 95°C for 15 sec
  2. 95°C for 15 sec
  3. 60°C for 1 min
  4. 95°C for 15 sec (melting curve from 60°C-95°C, read every 0.3°C); additional plate read at end

Ø  END

  1. Save experiment prior to running
  2. Make sure samples are in and door is closed. Go to “Run” – click “start run” to start program.
  3. When run is completed, analyze data.

·  Check melting curves and amplification plots.

·  If samples need to be omitted, right-click on them, select “Omit”, and then hit the “analyze” button.

·  If samples need to be included, select “Include”, and then hit the “analyze” button.

·  To export data, click “export” – browse to select desired file to export to. Export file type is excel (.xls)

Updated by RK 2.8.11