Measuring dsDNA concentration using the Nanodrop
Introduction
Materials
- Equipment
- Nanodrop
- Kimwipe Lint-Free task wipers
- Micropipette (p2)
- Pipette tips
- Reagents
- Purified DNA samples
- Elution buffer or alternative
Procedure
Important information to be documented throughout this procedure
- Samples measured
- If different from 1µL, volume used per read
- Concentration values reported by reads
- The location of project file, and the project name
- Unexpected and expected observations and results (either in writing or in picture form)
- Clarification notes (protocol modifications, issues, significant notes)
Important notes
- The default volume to scan is 1µL. The protocol assumes all volumes are 1µL.
A different volume may be used if desired. The nanodrop can read samples from 0.5 - 2µL.
- Prepare the proper solution to blank the nanodrop: Use the buffer or solution used to elute your sample.
- If results look extremely strange, or impossible (negative values), try to load your blank on the pedestal again and reblank.
Procedure:
Preparing the nanodrop
- Start the nanodrop laptop and ensure the USB cable to the nanodrop is connected.
This makes sure there are no other samples from a prior use.
- Finger vortex your samples and spin down with a tabletop centrifuge.
The DNA sample may be collected at the bottom of your tube. This can lead to inaccurate results on the nanodrop. This is especially important if analyzing a sample that was just eluted from a centrifugation step.
- Open the Nanodrop2000 software. Click "Nucleic Acid" in the main menu.
- If the program asks if you want to append new data to an existing project, click "No".
- Let the routine wavelength verification run with the arm down.
- Wipe the pedestal and the arm with a kimwipe. Set aside the kimwipe to use again.
The same kimwipe will be used for the duration of the protocol.
- To the top right of the interface, confirm that the sample Type is DNA. Change to DNA if necessary.
Blank Reading
- Load your blank on the pedestal and lower the arm carefully.
- Press "Blank".
No results will be shown, but the measure button will now be lit up.
- After scanning, wipe the pedestal and the arm with a dry section of the kimwipe.
Sample Reading
- Before scanning your sample, write the name of your sample in the "Sample ID" field to the top right.
PAUSEThe first sample you scan will trigger saving the workbook. Navigate to Desktop -> iGEM -> Training and save your file here after changing the name. Add in your name to the file name to differentiate your file from others.
- Load your sample and press "Measure".
- Lift the arm, and wipe the pedastal and the arm with a dry section of the kimwipe.
- Repeat step 20 to 22 as necessary.
Use a different blank if the samples used a different elution buffer.
- Go to the Report section to look at all your data.
In the report section, you can change Sample ID names.
Storage + Disposal
- Any DNA sample to store should be stored at -20C.
Cleanup
- Exit out of the program.
- Wet an unused section of the Kimwipe with DI Water. Wipe down both the pedestal and the arm.
- Dispose of Kimwipe
This protocol describes how to use the nanodrop to test concentration of double-stranded DNA fragments or plasmids.