Luckau Protocols

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(Tara Luckau Protocols)
(NanoDrop)
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====Purpose====
====Purpose====
The NanoDrop is used to quantify genetic material.
The NanoDrop is used to quantify genetic material.
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====Protocol====
# Assemble cafeteria tray
# Assemble cafeteria tray
#* Kim Wipes
#* Kim Wipes
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#* samples
#* samples
# The NanoDrop is located in North Life Sciences, room 325A
# The NanoDrop is located in North Life Sciences, room 325A
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#*  
+
#*[[Image:NanoDrop1000.jpg]]
 +
# Open the NanoDrop software, "ND1000" on the desktop
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# Choose "Nucleic Acids"
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# Initialize the instrument
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#* Place 2µL of NanoPure water on the pedestal
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#* Press OK
 +
#* when it's done, wipe pedestal and top connector with Kim Wipe
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# Calibrate the instrument
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#* Place 2µL of elution buffer on the pedestal
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#* For Clark's lab, elution buffer is 10mM Tris-Cl, pH 8.4
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#* Click "Blank"
 +
#* when it's done, wipe pedestal and top connector with Kim Wipe
 +
# Measure sample
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#* Place 2µL of sample on the pedestal
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#* Enter sample ID
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#* Click "Measure"
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#* wipe pedestal and top connector with Kim Wipe after each sample
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#* Re-calibrate the instrument each 10 samples, or so, by going back to step 6
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# Save data
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#* Click "Show Report"
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#* Click "Report", "Export Data"
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#* Click "Table Report"
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#* save to your USB drive, as "YYYYMMDD_NanoDrop", use "A" "B" "C", etc to designate multiple saves in the same day
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# Re-initialize the instrument each 30-50 samples, by going back to step 3
===Agarose Gel===
===Agarose Gel===

Revision as of 16:24, 3 November 2010

Contents

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
    • 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
  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
  • 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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