Protocol for usage of the Thermo Scientific NanoDrop 2000c Spectrophotometer

Goal:

Use the Jenkins lab NanoDrop at URI to measure the nucleic acid concentration and quantify purity ratios for a high molecular weight DNA sample from Acropora pulchra sperm.

Materials and Equipment:

• NanoDrop 2000c Spectrophotometer
• NanoDrop 2000c Software
• Gloves
• Kimwipe
• 0.1 uL to 2.0 uL pipette
• 0.1 uL to 2.0 uL filtered pipette tips
• Blank for measurement calibration (e.g., UltraPure water, Elution Buffer, etc. whatever your sample is eluted in)

Resources:

NanoDrop 2000c Manual

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Protocol Steps

1. Contact Dr. Bethany Jenkins or Dr. Chris Lane lab members to confirm that you can use the NanoDrop. There is also another NanoDrop in the URI RI-INBRE lab for free use.
2. Once trained on the Jenkins Lab NanoDrop, begin your processing by pressing the enter button a few times on the keyboard of the ViewSonic windows computer to view the screen (never turn off monitor or system, it is always on idle).
3. Double-click on the NanoDrop 2000 software on the desktop.
4. While waiting for the software to load, raise the metal grey sampling arm of the NanoDrop carefully. You will see a black ring with a small metal grey point in the center, this is the lower measurement pedestal, the upper measurement pedestal is a small grey metal point on the top of the raised sampling arm.

1. Using a kimwipe, gently wipe the lower and upper measurement pedestal.

1. After wiping the pedestals, you will see the software has loaded to a homepage that has a range of selections from Nucleic Acid to Protein BCA to Cell Cultures protocol options.

1. For my goals, I selected the Nucleic Acid protocol. Once you select this, it will ask you if you want to load the last workbook but you will select no so that you do not overwrite other work or save your work to someones workbook.

1. A box will then pop up in the program to run routine verification of measured wavelengths and you will hit enter or press okay. It will then take a few seconds and perform the routine verification process.

1. It will then bring you to a screen with an empty graph and in the top left it will say “Load your blank solution and press the blank button”

1. You will then use your filtered tips and 0.5 - 2uL pipette to pipette your blank (either the elution buffer you eluted your DNA in or Tris if you cleaned your product before, blank solution must match what your DNA is eluted in!) and load 2 uL of the blank onto the lower measurement pedestal.

2. Gently open the sampling arm and then clean upper and lower measurement pedestal again with a kimwipe. Pipette 2 uL of your blank solution and using your non-dominant hand, place the pipette on it for stabilization. Then gently dispense the blank onto the middle portion of the lower measurement pedestal to cover the small hole in the middle of the metal probe.

1. Gently lower the sampling arm (if you lower to fast an air bubble or splashing of your sample could cause issues).

1. In the top right corner of the screen, make sure to set your sample ID type to whatever you are measuring (e.g.,, DNA, RNA, ssDNA, etc.). I selected DNA for my goals.

1. Select the blank button on the top left side of the screen in the program.

2. Repeat steps 10-12 with your sample. The software will have a “Load your sample and press the measure button” command on the top left side of the graph.

1. Once your sample is loaded, press the measure button on the top left side of the screen that has a green play button icon. The software will then read your sample.

1. Once it is finished, it will ask you if you want to save the file, you can save to Kristinas folder on the documents tab if you would like but label with your initials and delete it after you take a photo. There is no internet on this computer so you cannot email it to yourself.

1. You will see a screen with a plot of the wavelength (nm) on the x axis, and 10 mm Absorbance on the y axis. You will also see your quality ratios and concentrations of your DNA. The concentrations in ng/uL read on a NanoDrop are will read much higher than the Qubit because it is not as selective for what it considers DNA and also detects contaminants and impurities.

1. You can use these resources to look into the meaning of your results further, but for DNA you usually want A280/260 ratios of 1.8-2.0 and A260/230 of > or equal to 1.8 for good purity.

Interpret NanoDrop Results

Interpreting NanoDrop Results

1. When you are finished, clean up your materials and clean the sampling arm upper and lower measurement pedestals with a kimwipe and exit out of the NanoDrop software. Do not turn off the computer!