qPCR - Initial Primer Tests with Positive Control Serial Dilutions

qPCR
CFX Connect
2023
Author

Sam White

Published

November 19, 2023

Intro

After quantifying the DNA and awaiting a response on how to handle the lack of DNA in 20 of the samples, I decided to create a serial dilution of some pooled DNA and test the three primer sets. I’d like to get a sense for their specificity, as well as their sensitivity.

All primers had been previously ordered/prepped by Tanya Brown. It’s my understanding that all three primer set sequences have been taken directly from previously published articles. One caveat is that those articles used these primer sets along with a probe oligo. Due to costs/time, these qPCRs (and all subsequent qPCRs) were performed using SYBR green (i.e. no probe oligo).

Serial Dilutions

Created a 10ng/uL (10,000pg/uL) “stock” consisting of three samples, two non-bleached (NB) and one bleachd (B). Selected the following samples and pooled them in the following fashion:

Samples Used

SAMPLE CONCENTRATION(ng/uL) VOLUME_USED(uL) DNA(ng)
T4.M.41.NB 21.6 3 64.8
T5.M.74.B 10.9 3 32.7
T4.M.10.NB 4.44 3 13.32
TOTAL 9 110.82

Dilution Calculations

Created the 10,000pg/uL stock solution:

Concentrationinitial(ng/uL) Volumeinitial(uL) Concentrationfinal(ng/uL) Volumefinal(uL) Volume H2O to reach Volumefinal
12.31333333 9 10 11.082 2.082

10-fold serial dilutions were created, down to 10pg/uL, using 2uL from each previous “stock” in 18uL of H2O.

qPCRs

All qPCRs were run in triplicate for each dilution (1000pg, 100pg, 10pg), with SsoAdvanced Universal SYBR Green Supermix in 20uL reactions. Each primer was utilized with an initial “working stock” concentration of 10uM and at a final concentration of 0.25uM in each qPCR reaction. See the qPCR Report for cycling parameters.

qPCR calculations are here:

Results

Output files

Raw qPCR data (.pcrd; Requires CFX Maestro):

qPCR data (CSV):

qPCR Report (PDF):

CAct Amplification

Amplification Plot Melt Plot
CAct Amplification plot showing a single sample amplification of 100pg input DNA at Cq ~= 39 CAct melt plot showing a single melt curve

Here we see a single sample amplifying, which corresponds to 100pg of input DNA. It’s odd there’s no amplification in any of the 1000pg samples, considering we got late amplification in one of the 100pg samples… Will have to re-run. Possibly will order fresh primer stocks.

DAct Amplification

Amplification Plot Melt Plot
DAct Amplification plot showing sample amplification of 1000pg (yellow lines) and 100pg (pink lines) of input DNA DAct melt plot showing melt curves of 1000pg (yellow lines) and 100pg (pink lines) of input DNA

We see all three reps of 1000pg DNA amplifying (yellow lines) and two of the three 100pg reps amplifying (pink lines). No amplification with 10pg of input DNA. Melt curves look good.

Mcap_PaxC Amplification

Amplification Plot Melt Plot
Mcap_PaxC Amplification plot showing sample amplification of 1000pg (yellow lines) and 100pg (pink lines) of input DNA Mcap_paxC melt plot showing melt curves of 1000pg (yellow lines) and 100pg (pink lines) of input DNA

We see all three reps of 1000pg DNA amplifying (yellow lines) and all three 100pg reps amplifying (pink lines). No amplification with 10pg of input DNA. Melt curves look good.

SUMMARY

DAct and Mcap_PaxC primers appear to work well and produce amplification at 1000pg and 100pg.

CAct primer set only amplified a single sample at 100pg. Will need to repeat this, but will plan on ordering a fresh primer set to ensure that issues aren’t due to age.

References

Cunning, Ross, and Andrew C. Baker. 2012. “Excess Algal Symbionts Increase the Susceptibility of Reef Corals to Bleaching.” Nature Climate Change 3 (3): 259–62. https://doi.org/10.1038/nclimate1711.
Cunning, R, R Ritson-Williams, and RD Gates. 2016. “Patterns of Bleaching and Recovery of Montipora Capitata in Kāne‘ohe Bay, Hawai‘i, USA.” Marine Ecology Progress Series 551 (June): 131–39. https://doi.org/10.3354/meps11733.