Jan 27, 2025

Public workspaceQuantifying and Checking Genomic DNA V.2

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvr46epgmk/v2
Quantifying and Checking Genomic DNA
  • Carlos Goller1,
  • Carly Sjogren1
  • 1[North Carolina State University]
Nidhi Grover
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvr46epgmk/v2
Protocol Citation: Carlos Goller, Carly Sjogren 2025. Quantifying and Checking Genomic DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr46epgmk/v2Version created by Carlos Carlos Goller
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: January 27, 2025
Last Modified: January 27, 2025
Protocol Integer ID: 119086
Keywords: DNA, assessment, fluorometer, nano spectrophotometer, checking genomic dna overview, bacterial isolate, dna sample, dna quality, size of the dna quality, dna concentration, dna quantity, dna yield, implen nano spectrophotometer, agilent tapestation electrophoresis system, genomic dna, lab safety, dna, gel electrophoresi, nano spectrophotometer, following lab skill, qubit fluorometer, lab, lab skill
Funders Acknowledgements:
NC State Biotechnology Program
Abstract
Overview and Goals
Your bacterial isolate has been grown on agar plates and lysed open, and genomic DNA has been isolated for sequencing. Next, we need to quantify our DNA yield and assess its integrity. We will use the IMPLEN nano spectrophotometer and Qubit to measure our DNA quantity. We will also use an Agilent TapeStation electrophoresis system to assess the size of the DNA quality.

Learning Objectives
After completing this lab, you will gain the following lab skills:
  • Lab safety and proper personal protective equipment (PPE)
  • Proper use of a nano spectrophotometer and Qubit fluorometer to analyze DNA samples.
  • DNA concentration and integrity analysis using absorbance, fluorescence, and gel electrophoresis.
Image Attribution
Image purchased from The Noun Project and recolored by Carlos C. Goller
Materials
  • One 10 µL micropipette (p10)
  • Filtered tips for p10
  • Kimwipes
  • Tip disposal container
  • Distilled water
  • New England BioLabs (NEB) gDNA Elution buffer (or whatever buffer experimental DNA is stored in)
  • IMPLEN NP80 nano spectrophotometer
  • Qubit High Sensitivity (HS) 1X working solution and Qubit fluorometer
  • TapeStation Genomic DNA reagents
  • Your genomic DNA samples
Troubleshooting
Before start
Review the protocols and figures below to learn about how the IMPLEN nano spectrophotometer, Qubit fluorometer, and Agilent TapeStation electrophoresis systems work.

Activity 1: Quantification of DNA with a Nano Spectrophotometer
In the lab, we will use the IMPLEN NP-80 nano spectrophotometer to measure the concentration and assess the purification of your DNA.

Quantification of DNA with Nano Spectrophotometers. Diagram of DNA fragments in amicrofuge tube and three points: spectrophotometer, DNA concentration, and potential RNA and protein contamination. On the right, a nano spectrophotometer diagram with two OD (y-axis) an lambda (x-axis) graphs. The top one has a peak at 260 nm for nucleic acids; the bottom one has a peak wavelength of 280, suggesting protein contamination.


Before starting, review these two documents:
Nucleic Acid Spectrophotometer and

IMPLEN Technical Note 12 – Best Practice for Nucleic Acid Quantifications
Wipe the pedestal carefully with a damp wipe.

If the device is off, initialize the instrument by pressing the power button on the back left.

The machine will initialize and may make some noises; do not be alarmed.
Wait for the instrument to initialize and display the menu on the touchscreen.

Clean the pedestal one more time with a damp wipe.
Blank with the buffer or water used to elute your DNA.
Once the machine has been initialized, lift the bar from the pedestal and wipe each side with a Kimwipe to ensure it is clean and dry.
Wash
Next, you will need to load a “blank” sample of buffer. This is to calibrate the machine to the particular buffer in which your DNA is housed.
Using a p10, addAmount2 µL of your DNA solvent to the lower pedestal (review image below) and gently lower the nano spectrophotometer arm.

Pipetting
On the computer screen, select “Blank” on the screen after loading.
Next, measure your blank by selecting “Sample” on the screen. You can enter a name such as "Blank C6 Buffer."

The results of the blank should come back near 0 for each metric. If not, clean off the pedestal thoroughly and then load and measure another blank. Once you’ve successfully measured your blank, you may measure your sample.
Measuring each of your samples.
Using a 10 µl filtered pipette tip, add Amount2 µL of your sample to the center of the pedestal.

Note
Do not fully depress the pipette when adding the sample to the pedestal. Any air bubbles in the drop can affect the measurement.

Make sure the drop is on or close to the center of the pedestal. You can use the pipette tip to slowly move the drop around if you misplace it.

Close the bar, and then look to make sure the droplet is touching both the upper and lower measurement pedestals as on the right.
After ensuring the sample is properly placed, press measure in the top left corner of the screen.

Note
You’ll hear a click from the machine after starting the measurement - that’s good.

For each read, record the ng/µl and 260/280 values in your spreadsheet, in addition to whatever other information your instructor requests.
Critical
Once the measurement has been taken and data recorded, open the bar and wipe off each side of the pedestal with a Kimwipe.
Safety information
This is crucial to ensuring accurate and consistent results. Two or three wipes on each side is fine, but be sure it is dry each time before moving onto the next sample.

Wash
Only after cleaning the pedestal can you start to add another sample.
After finishing your last read, clean the pedestal for the next user.
Wash
Recording you DNA quantifications
Create an Excel or Google spreadsheet to keep track of your data from the nano spectrophotometer. Title it with the date and the name of your samples. Record the following values:
  1. The concentration of your DNA in ng/µl,
  2. The ratio of the absorbance at 260 and 280. DNA should have a ratio of 1.8; if RNA is present, the ratio will be higher. If protein is present, the ratio will be lower than 1.8.
  3. 230/260 ratio that is used to learn about other substances that may be in your sample
Analyze
Activity 2: DNA Quantification with Qubit Fluorometer
2m
Quantification of DNA with the Qubit Fluorometer. Diagram of DNA fragments next to microfuge tube bound by green dye. Three key points are stated: fluorometer, accurate DNA concentration, and high sensitivity with fluorescent dye. A diagram of a Qubit fluorometer is on the right of the image.
Follow the link, scroll to the bottom of the webpage, and watch this video about the Qubit fluorometer: Qubit™ 4 Fluorometer video (4:29 min)


Note
The Qubit™ 1X dsDNA HS Assay requires two standards. We use thin-wall, clear, 0.5-mL PCR tubes (Cat. No. Q32856) for the Qubit™ 4 Fluorometer.

Label the tube lids with a Sharpie. Label 3 tubes (2 for standards and 1 for your sample):
  • "1" (standard)
  • "2" (Standard)
  • Bacterial species number. For example: Isolate 5.


Safety information
Do not label the side of the tube as this could interfere with the sample read.


Prepare your standards to calibrate your sample measurement:
  • Each standard tube requires 190 µL of Qubit™ working solution
  • Add Amount10 µL of each Qubit™ standard to the appropriate tube.
  • The final volume in each tube must be Amount200 µL

Prepare your sample:
  • Each sample tube requires Amount195 µL of Qubit™ working solution
  • Add Amount5 µL of each user sample to the appropriate tube using a p10.
  • The final volume in each tube must be Amount200 µL


Pipetting
Mix each sample by vortexing for Duration00:00:05 .

5s
Allow all tubes to incubate at TemperatureRoom temperature for Duration00:02:00 .
Safety information
Use a timer and keep the samples protected from light.


2m
Incubation
Calibration of the Qubit™ Fluorometer requires the standards to be inserted into the instrument in the right order. Read the prompts from the Qubit to insert the standards in the correct order. Note: we will assist you through this process. The information provided below is so you can review the entire process.
  • On the Home screen of the Qubit™ Fluorometer, press the 1X dsDNA High Sensitivity (HS) assay icon. The “Read standards” screen is displayed. Press Read Standards & run samples to proceed. Note: If you have already performed a calibration for the selected assay, the instrument prompts you to choose between reading new standards and running samples using the previous calibration. If you want to use the previous calibration, press Run samples and skip to step 2.4.
  • Insert the tube containing Standard #1 into the sample chamber, close the lid, then press Run standards. When the reading is complete (~3 seconds), remove Standard #1.
  • Insert the tube containing Standard #2 into the sample chamber, close the lid, and then press Read standards. When the reading is complete, remove Standard #2. The instrument displays the graphical results on the Standards complete screen.
  • Press Next from the Standards complete screen to read your Samples.
  • In the Sample volume screen, enter the sample volume added to the assay tube (from 1-20 µL). Enter the volume directly in the sample volume text box, use the + or – buttons or adjust the sample volume wheel to select the sample volume added to the assay tube. Note: The sample volume used (1-20 µL) changes the assay accuracy range. A different sample volume or assay may be required if the sample concentration is outside of what the assay can accurately quantify.
  • Insert a sample tube into the sample chamber, close the lid, then press Run sample. When the reading is complete (~3 seconds), remove the sample tube.
Record your concentrations in ng/µl in your notes and directly on the tubes of your genomic DNA samples.
Activity 3: Genomic DNA Assessment with TapeStation
32m

The Agilent TapeStation is an automated gel electrophoresis system that allows users to separate nucleic acids to obtain information about their size and distribution.
Work with your instructors to prepare samples for analysis.
Allow the Genomic DNA Reagents to equilibrate at room temperature for Duration00:30:00 .

30m
Flick the Genomic DNA ScreenTape device and insert it into the ScreenTape nest of the TapeStation instrument.
  • Select required sample positions in the TapeStation Controller software. The required consumables (tips, further ScreenTape devices) are displayed in the TapeStation Controller software.
Vortex reagents and samples. Spin down before use.
Prepare ladder:
  • Pipette Amount10 µL Genomic DNA Sample Buffer and Amount1 µL Genomic DNA Ladder at position A1 in a tube strip.

Pipetting
For each sample, pipette Amount10 µL Genomic DNA Sample Buffer and Amount1 µL DNA sample in a tube strip

Pipetting
Apply caps to tube strips .
Mix liquids using the IKA MS3 vortexer at Centrifigation2000 rpm for Duration00:01:00

1m
Spin down samples and ladder for Duration00:01:00

1m
Sample Analysis
  • Load samples into the TapeStation instrument.
  • Place ladder in position A1 on the tube strip holder.
  • Carefully remove caps of tube strips.
  • Visually confirm that liquid is positioned at the bottom.
  • Click Start.
  • The TapeStation Analysis software opens automatically after the run and displays results.

Note
Critical Thinking Questions for Quantifying and Checking Genomic DNA
  1. Before you measure your DNA sample using the nano spectrophotometer, you blank it using the dissolved solvent in which your DNA is dissolved.
  2. What do you think the impact of using the wrong solvent could be? For example, water can be usedinstead of TE buffer.
  3. What would happen if you "blanked" the nano spectrophotometer using your DNA sample? Would you be able to detect your DNA measurement?
  4. The nano spectrophotometer uses photospectrometry to measure your sample, while the Qubit uses fluorescence activity from chemicals that can bind DNA. What do you think are some advantages of using each method?
  5. Compare your data from the nano spectrophotometer and the Qubit: are they similar or different quantifications? If they are different, which would you trust and why?
  6. From the TapeStation, we will receive a DNA integrity number (DIN). What does a higher (for example, 9) or lower value (for example, 4) tell you about your DNA?



Acknowledgements
We appreciate the support from the NC State Biotechnology Program and students who have helped improve this and other protocols.