Luckau Protocols:NanoDrop

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NanoDrop Protocol

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

FYI

• 260/280 = ratio of sample absorbance at 260 and 280nm, used to assess the purity of DNA and RNA; 1.8 is "pure" for DNA, <1.8 may indicate presence of contaminants
• 260/230 = ratio of sample absorbance at 260 and 230nm, used as a secondary measure of nucleic acid purity; 1.8-2.2 is "pure", <1.8 may indicate presence of contaminants
• NanoDrop User's Manual Media:NanoDrop1000-v3.7-UsersManual.pdf