Oct 05, 2025

Public workspaceMeasure DNA concentration

DOI
https://dx.doi.org/10.17504/protocols.io.36wgqpeb3vk5/v1
  • igemnthuth 1
  • 1iGEM_NTHU_Taiwan
  • iGEM NTHU
igemnthuth
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgqpeb3vk5/v1
Protocol Citation: igemnthuth 2025. Measure DNA concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqpeb3vk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 03, 2025
Last Modified: October 05, 2025
Protocol Integer ID: 228966
Keywords: measure dna concentration measure the dna concentration, measure dna concentration measure, purity of the purified pcr product, dna concentration, purified pcr product, pcr, spycatcher segment, dna, spycatcher, purity
Abstract
Measure the DNA concentration and purity of the purified PCR product (SpyCatcher segment) to ensure it meets Sanger sequencing submission requirements, and dilute or concentrate as needed.
Materials
  • Purified PCR product (product purified in the previous step using a DNA purification kit)
  • Nuclease-free water
  • 70% ethanol and Kimwipes
  • NanoDrop spectrophotometer
  • Pipettes and sterile tips
Troubleshooting
Starting and Calibrating the Nanodrop
Turn on the Nanodrop device and launch the software.
Select Nucleic Acid → dsDNA mode.
Pipette 2 µL of nuclease-free water onto the lower measurement pedestal.
Close the arm and click Blank to calibrate the background.
Measuring DNA Sample Concentration
Using a clean pipette tip, pipette 2 µL of the purified PCR sample onto the lower pedestal.
Close the arm and click Measure.
ecord the following data:

  • Concentration (ng/µL) — typically 10–20 ng/µL is required for sequencing.
  • 260/280 ratio (purity) — should be approximately 1.8–2.0.
Clean both the upper and lower pedestals with 70% ethanol and a Kimwipe.
Diluting DNA Concentration if Necessary
If the DNA concentration is too high (>100 ng/µL), dilute to 10–20 ng/µL using the following formula:

V1​×C1​=V2​×C2​
Add nuclease-free water to dilute.
Mix thoroughly and re-measure with the Nanodrop to confirm the correct concentration.