Qubit DNA Quantification

SIFP_2025
Author

Kathleen Durkin

Published

July 14, 2025

Used Qubit flourometer to quantify DNA concentration of extracted E. tourneforti samples.

LAB 1X dsDNA Qubit Protocol

Note: Used 2uL of sample DNA for each Qubit rxn

Results

Had to assign new labels to the samples for Qubiting, since the USNM Catalog numbers are too large to fit on the Qubit tube lids

Standards (first run):
Standard #1: 37.57
Standard #2: 14064.10

Standards (second run, with dilutions):
Standard #1: 36.17
Standard #2: 14557.00

Year.Collected Catalog.Number Qubit.label Concentration.ngul
1886 USNM 14366 A01 * 0.173
1886 USNM 14399 A02 33.9
1898 USNM 19054 A03 * 6.03
1898 USNM 42137 A04 15
1960 USNM 51730 A05 20.9
1960 USNM 51732 A06 89.4
1960 USNM 51728 A07 35.2
1960 USNM 51892 A08 86.3
1912 USNM 52295 A09 * 7.08
2000 USNM 100609 A10 161
2000 USNM 100610 A11 84.7
2000 USNM 1007393 A12 116
2002 USNM 1018355 A13 44.3
1912 USNM 1180630 A14 * 1.41
2019 USNM 1606824 A15 167
2019 USNM 1606826 A16 189
2019 USNM 1740336 A17 163
2019 USNM 1740363 A18 152
2019 USNM 1740390 A19 126
2019 USNM 1740407 A20 133
1880 USNM 50368 B01 26.5
1912 USNM 50603 B02 * 2.28
1960 USNM 51727 B03 27
1960 USNM 51729 B04 48.9
1960 USNM 51857 B05 43.7
1960 USNM 51858 B06 28.5
1960 USNM 51859 B07 43.9
1960 USNM 51860 B08 56
1960 USNM 51861 B09 52
Note

Bolded values indicated the sample was originally too high concentration to be accurately quantified by the Qubit assay. For these samples I made a 10X dilution (1uL DNA sample, 9uL Zymo DNA Elution Buffer), then re-Qubited the dilutions. Values shown above are the calculated “true” concentrations (10X dilution concentration, multiplied by 10).

Generally, almost all extractions have a good amount of DNA! The recommended minimum quantity of DNA for Nanopore Native Library Prep is 400ng. Since I’ll have ~40uL DNA left for each sample after eluting in 50uL and using a small portion for QC steps, I’m aiming for a minimum DNA concentration of 10 ng/uL.

Only 5 samples failed to meet this threshold (starred above). All 5 are from my set of old specimens (>100 yo). I only have 8 total specimens from this collection period, and I want an n=5 for sequencing. I’ll need to do additional extractions for the 5 specimens with low concentrations (USNM 14366, USNM, 19054, USNM 52295, USNM 1180630, USNM 50603). I may also want to do an additional extraction for USNM 42137 (15.0 ng/uL), since its not much higher than my desired minimum concentration.

library(googlesheets4)
Warning: package 'googlesheets4' was built under R version 4.2.3
library(ggplot2)
Warning: package 'ggplot2' was built under R version 4.2.3
conc <- read_sheet("https://docs.google.com/spreadsheets/d/1zDMy7AOCM8Ug85q4vr9CoMDxfF-zDJk1DnaxakSt1Hg/edit?usp=sharing")
! Using an auto-discovered, cached token.
  To suppress this message, modify your code or options to clearly consent to
  the use of a cached token.
  See gargle's "Non-interactive auth" vignette for more details:
  <https://gargle.r-lib.org/articles/non-interactive-auth.html>
ℹ The googlesheets4 package is using a cached token for 'kdurkin1@uw.edu'.
✔ Reading from "E_tourneforti".
✔ Range 'E_tourneforti_samples'.
ggplot(conc, aes(x=Year.Collected, y=Concentration.ngul, color=Country)) +
  geom_point(size=2) + 
  geom_line(aes(y=10), color='black') +
  theme_minimal()
Warning: Removed 3 rows containing missing values or values outside the scale range
(`geom_point()`).

(anything collected in the US was collected off the coast of Florida)

Horizontal line indicates the 10 ng/uL cutoff, or the minimum concentration that will yield at least the library prep requirement of 400ng DNA from a volume of 40uL.