Wednesday, November 18, 2015


Objective: Take cDNA and perform a qPCR to quantify gene expression for 3 sets of samples amplified using 2 different primers


qPCR Protocol

Standard Operating Protocol (SOP)

Written 20150702 by Sam White.
  • SsoFast EvaGreen Supermix (BioRad: 172-5203)
  • Primer working stocks (10uM)
  • DNase-free H2O (NanoPure H2O)
Personal Protective Equipment (PPE):
  • Gloves


Total Time: ~ 2.0 - 4.0hrs
Cost/sample: ~ $0.42
  1. Read the manufacturer's protocol.
  2. Read this protocol.
  3. Verify sufficient quantities of reagents and samples before beginning.
  4. Wear clean gloves.
  5. Prepare master mix. Be sure to make a master mix volume that will accommodate the following: all of your samples, two water (i.e. no template controls; NTC) samples, plus an extra 10% to accommodate pipetting errors. A single reaction is shown below:
    ComponentVolume(uL)Final Concentration
    2x SsoFast EvaGreen Supermix101x
    Forward Primer (10uM)0.5uL0.2uM
    Reverse Primer (10uM)0.5uL0.2uM
    WaterVariableUse to bring reaction volume up to 20uL (including template)
  6. Distribute appropriate amount of master mix (volume of master mix + template = 20uL) to white PCR plate.
  7. Add template.
  8. Cap with optical PCR caps.
  9. Spin plate for 1min @ 3000g.
  10. Put plate in real-time PCR machine.
  11. Recommended cycling parameters (40 cycles) are listed below. They may need to be changed to accommodate your specific primers/samples. See manufacturer's protocol for recommendations.
    Step 1 - 98C 2mins
    Step 2 - 98C 5secs
    Step 3 - 60C 5secs
    Step 4 - Plate read
    Step 5 - Got to Step 2 39 more times
    Step 6 - Melt curve 65C - 95C, increment 0.5C, wait 2secs, plate read.



Sample Definitions: 
1-1, 1-2, 2-1, 2-2 Samples taken originally for sequencing work added them to compare to samples taken at a later date to check for variation due to time in captivity,

Day 1-4 are samples taken during manipulation experiment where animals were under artifical lighting to simulate day time hours.

Night 1-4 animals left in the dark for two hours to simulate a night time environment.


qPCR calculations

Primer set 1


Link to data:

Melting Curve

Primer Set 2


Melting Curve

Note: realized that I had forgotten my no template controls when doing the experiment will rerun qPCR to confirm that data is valid.

1 comment:

  1. Can you post text (not picture) of Sample ID and Ct value for Primer set 1?