(Prepare this in your own lab):

Designing an Absolute Quantification Experiment

Typical AQ exoerunebts are designed for traditional (singleplex) PCR, where a single primer pair plus a TaqMan proble or SYBR binding dye are present in a reaction. The following sections describe decisions required for AQ experiments.

• Specifying the Components of an AQ Experiment

  For each AQ experiment, specify:

  • An unkown - The nucleic acid sequence that you are quantifying.
  • Standards - This guide assumes that you have generated a set of standards for each target sequence that you are quantifying.
  • Replicate wells - For absolute quantification assays, use three or more replicate reactions per sample to ensure statistical significance.
• Selecting the Chemistry

  (select 1 or 2):

  1. TaqMan Reagents or Kits
  2. SYBR Green Reagents
• Selecting One- or Two-Step RT-PCR
  • One-Step: Perform RT and PCR in a single reaction.
  • Two-Step: Perform RT and PCR in separate reactions.
• Converting RNA to C-DNA
  1. Isolating Total RNA
  2. Quality of RNA: have an A260/280 greater than 1.9, be intact wehn visulaized by gel electrophresis, not contain RT or PCR inhibitors.
  3. Starting Concentration of total RNA should be 0.1-10 ug.
  4. Reverse Transcription (1x):
     • 10 ul 10 x RT Buffer
     • 4 ul 25x dNTPs
     • 10 ul 10x random primers
     • 5 ul MultiScribe Reverse Transcriptase
     • 21 ul Nuclease-free water
     • Total: 50 ul
     • 25 C for 10 min, then 37 C for 120 min.
     • Store all cDNA samples at -15 to -25 C.
• Running an AQ Plate
  1. Preparing the PCR Master Mix
  2. Preparing the Reaction Plate

Take your plate to:

33 Life Science Building
Auburn University Genomics and Sequencing Laboratory

Next Topic: Setting up ABI 7500 Real Time PCR Systems