Luckau Protocols:NanoDrop

NanoDrop Protocol

Tara K. Luckau's Home Page Conservation Genetics Lab Notebook Tara's Protocols

Purpose

The NanoDrop is used to quantify genetic material. Although there are many different methods to quantifying DNA, the NanoDrop is a spectrophotometer that calculates absorbance of a sample across different wavelengths.

Protocol

1. Assemble cafeteria tray
   • Kim Wipes
   • 2µL pipette
   • pipette tips
   • gloves
   • USB drive
   • water and buffer tubes
   • samples
2. The NanoDrop is located in North Life Sciences, room 325A
   • 
3. Open the NanoDrop software, "ND1000" on the desktop
4. Choose "Nucleic Acids"
5. Initialize the instrument
   • ensure upper and lower pedestal surfaces are clean by wiping with Kim Wipe
   • Place 2µL of NanoPure water on the lower pedestal
   • Lower the sampling arm and press OK
   • when it's done, wipe upper and lower pedestals with Kim Wipe
6. Calibrate the instrument
   • Place 2µL of elution buffer on the pedestal
   • For Clark's lab, elution buffer is 10mM Tris-Cl, pH 8.4
   • Click "Blank"
   • when it's done, wipe upper and lower pedestals with Kim Wipe
7. Measure sample
   • Place 2µL of sample on the pedestal
   • Enter sample ID
   • Click "Measure"
   • wipe upper and lower pedestals with Kim Wipe after each sample
   • Re-calibrate the instrument each 10 samples, or so, by going back to step 6 and clicking "Re-Blank" instead of "Blank"
8. Save data
   • Click "Show Report"
   • Click "Reports", "Save Report"
   • Click "Export Report Table Only"
   • save to your USB drive, as "YYYYMMDD_NanoDrop", use "A" "B" "C", etc to designate multiple saved files in the same day
9. Re-initialize the instrument each 30-50 samples, by going back to step 3
   • remember to save your data before your re-initialize
10. Clean up thoroughly! Other people use this instrument!
11. don't forget to enter data into spreadsheet

Purity Assessment

• ratio of sample absorbance at 230, 260 and 280nm is used to assess sample purity. The ratios and information relevant to our work with DNA is given below:

260/280 Ratio

• used to assess purity of DNA and RNA
• pure DNA: ~1.8
• <1.8 → residual reagent form extraction, low nucleic acid concentration (<10 ng/µL)
• >1.8 → not an issue!

260/230 Ratio

• used as a secondary measure of nucleic acid purity
• pure nucleic acid: 2.0-2.2
• <2.0 → residual guanidine
• >2.2 → blanked on dirty pedestal, inappropriate blank (should be same pH and ionic strength)

Resources

• NanoDrop User's Manual Media:NanoDrop1000-v3.7-UsersManual.pdf
• NanoDrop Technical Bulletin T042: Assessment of Nucleic Acid Purity