Luckau Protocols

Tara Luckau Protocols

Clark Laboratory

NanoDrop

Purpose

The NanoDrop is used to quantify genetic material.

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
   • 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
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 saves in the same day
9. Re-initialize the instrument each 30-50 samples, by going back to step 3

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
• 

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