Amplification Curves


#1

Hello,

I just purchased the single channel qPCR machine and tried running some samples after calibration. However, I was unable to detect any amplification curves using the software. I used DNA and the Chai green master mix. The point of the qPCR is determine how many cycles I should run to amplify Illumina DNA libraries for sequencing. I thought I should have at least seen primer-dimer amplification but the plots were blank. Any ideas of what I may have done wrong?

Thank you!

Mix:

(1) 1µl of DNA Pre-Library
(2) 0.75µl of 10uM of indexing primer i5 (F primer)
(3) 0.75µl of 10uM of indexing primer i7 (R primer)
(4) 17.5 ul Chai Green Master Mix (2x)

Program: initial denature at 95°C for 5 min, the following (98°C for 20 s, 60°C for 15 s, 72°C for 30 s) for 30 cycles, final extension at 72°C for 5 min, hold at 4°C.


#2

Hi Jlindo,

Thanks for your question!

A couple things I would like to confirm with you:

  1. Is your final volume 20 uL? The Chai Green Master Mix is a 2X mix so there can be inhibition if you do not dilute it to 1X in the final reaction.

  2. Do you have the gather data step turned off at the end of the extension step? If this is turned off, you will not see any curves on the result screen since the instrument is running as a regular thermocycler at this point. Please see the attached screenshot for reference.

  3. Can you send me a screenshot of your protocol editor page, similar to my attached image?

  4. Do the Cq values display on the right side of the results screen?

  5. Can you send me a full screenshot of your amplification results page and your thermal profile graph? To access the thermal profile graph, please select the upside-down arrow next to “Amplification Curve” on the results screen. Then proceed to select the “Thermal Profile” graph.

Let me know if anything is unclear.

Cheers,
Lily


#3

Hi Lily,

I had the “Gather Data” options turned off. It works perfectly now.

Thank you!

John


#4

Hi John,

Glad to hear that fixed the issue! :slight_smile:

Cheers,
Lily