Luckau Protocols

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(Tara Luckau Protocols)
(Tara Luckau Protocols)
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[[Agarose Gel]]
[[Agarose Gel]]
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===NanoDrop===
 
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====Purpose====
 
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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.
 
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====Protocol====
 
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# Assemble cafeteria tray
 
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#* Kim Wipes
 
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#* 2µL pipette
 
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#* pipette tips
 
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#* gloves
 
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#* USB drive
 
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#* water and buffer tubes
 
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#* samples
 
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# The NanoDrop is located in North Life Sciences, room 325A
 
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#*[[Image:NanoDrop1000.jpg]]
 
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# 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
 
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#* 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"
 
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#* when it's done, wipe pedestal and top connector with Kim Wipe
 
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# 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 and clicking "Re-Blank" instead of "Blank"
 
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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 saved files in the same day
 
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# Re-initialize the instrument each 30-50 samples, by going back to step 3
 
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#* remember to save your data before your re-initialize
 
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# Clean up thoroughly! Other people use this instrument!
 
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# don't forget to enter data into spreadsheet
 
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===Agarose Gel===
 
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====Purpose====
 
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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.
 
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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.
 
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====Materials To Be Familiar With====
 
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* Gel Rig, caster tray, combs
 
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* [[Image:OwlA2Large.jpg]]  [[Image:OwlA2Large_draw.jpg|300 px]]
 
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====Protocol====
 
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# Make gel using 1x TAE and agarose
 
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#* Use 1% for genomic DNA, 2% for amplified DNA
 
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{| border="1" style="margin: 1em auto 1em auto" "text-align: center"
 
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|-
 
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! Gel Rig Size !! 1x TAE (mL) !! 1.5% agarose (g) !! 2% agarose (g) !! Gel Red (µL) !! Sample volume (µL) !! max voltage (V)
 
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|-
 
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| small || 50 || _ || 1 || 5 || 4-6 || 80
 
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|-
 
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| rowspan="2"| medium || rowspan="2"| 130 || rowspan="2"| _ || rowspan="2"| 2.6 || rowspan="2"| 13 || 24-tooth comb: 6-10 || rowspan="2"| 120
 
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|-
 
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| 36-tooth comb: 3-5
 
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|-
 
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| large || _ || _ || _ || _ || _ || 170
 
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|}
 
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#
 

Revision as of 19:07, 3 November 2010

Tara Luckau Protocols

Clark Laboratory

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

NanoDrop

Agarose Gel

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