Luckau Protocols

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Contents

Tara Luckau Protocols

Clark Laboratory

Following are the protocols used in Dr. Rulon Clark's laboratory.

NanoDrop

Agarose Gel

NanoDrop

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
    • Image:NanoDrop1000.jpg
  3. Open the NanoDrop software, "ND1000" on the desktop
  4. Choose "Nucleic Acids"
  5. Initialize the instrument
    • Place 2µL of NanoPure water on the pedestal
    • Press OK
    • when it's done, wipe pedestal and top connector 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 pedestal and top connector with Kim Wipe
  7. Measure sample
    • Place 2µL of sample on the pedestal
    • Enter sample ID
    • Click "Measure"
    • wipe pedestal and top connector 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 "Report", "Export Data"
    • Click "Table Report"
    • 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

Agarose Gel

Purpose

Agarose gels are used to verify the presence and estimate the size of DNA (both genomic and amplified). When loaded into a gel and subjected to an electric current, DNA fragments migrate to the positive terminal. Small DNA fragments migrate more rapidly than large ones. By running a commercially available ladder of fragments of known size, we can estimate the DNA fragment size in our sample of interest.

To visualize the DNA after electrophoresis, a dye called Gel Red is added during the agarose-making process. The dye binds to the DNA and flouresces when exposed to UV light.

Materials To Be Familiar With

  • Gel Rig, caster tray, combs
  • Image:OwlA2Large.jpg

Protocol

  1. Make gel using 1x TAE and agarose
    • Use 1% for genomic DNA, 2% for amplified DNA
Gel Rig Size 1x TAE (mL) 1.5% agarose (g) 2% agarose (g) Gel Red (µL) Sample volume (µL) max voltage (V)
small 50 _ 1 5 4-6 80
medium 130 _ 2.6 13 24-tooth comb: 6-10 120
36-tooth comb: 3-5
large _ _ _ _ _ 170
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