Luckau Protocols

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(Tara Luckau Protocols)
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==Tara Luckau Protocols==
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Clark Laboratory
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| width="600" style="background-color: #BCED91;" align="center"| <span style="font-size:32px;"> Tara's Protocols </span>
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Following are the protocols used in Dr. Rulon Clark's laboratory.
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|align="right" style="background-color: #9DB68C;" |
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<span style="font-size:16px;"> [[User:Tara_K._Luckau | Tara K. Luckau's Home Page]] </span>
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[[NanoDrop]]
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[[User:Tara_K._Luckau/Notebook/Team_ConGen | Conservation Genetics Lab Notebook]]
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[[Agarose Gel]]
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===NanoDrop===
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[[Luckau_Protocols | Tara's Protocols]]
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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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==Protocols used in Dr. Rulon Clark's laboratory==
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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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===Lab Processes===
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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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* [[Luckau_Protocols:NanoDrop | NanoDrop]]
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|-
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* [[Luckau_Protocols:PCR | PCR]]
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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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* [[Luckau_Protocols:Agarose Gel | Agarose Gel]]
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* [[Luckau_Protocols:FragmentAnalysisSubmission | Fragment Analysis Submission]]
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| small || 50 || _ || 1 || 5 || 4-6 || 80
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* [[Luckau_Protocols:ShipDryIce | Shipping with Dry Ice]]
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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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===Reagents===
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| 36-tooth comb: 3-5
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|-
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| large || _ || _ || _ || _ || _ || 170
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* [[Luckau_Protocols:Low TE | Low TE]]
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|}
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* [[Luckau_Protocols:Tris-Cl | Tris-Cl]]
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* [[Luckau_Protocols:KCl | KCl]]
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* [[Luckau_Protocols:MgCl2 | MgCl<sub>2</sub>]]
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* [[Luckau_Protocols:TAE | TAE]]
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* [[Luckau_Protocols:1Kb Ladder | 1Kb Ladder]]
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* [[Luckau_Protocols:dNTPs | dNTPs]]
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* [[Luckau_Protocols:PCR Buffers A-H | PCR Buffers A-H]]
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* [[Luckau_Protocols:PrimerResuspension | Primer Resuspension]]
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===Software Programs===
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* [[Luckau_Protocols:GeneMarker | GeneMarker]]
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* [[Luckau_Protocols:Scoring | How I Score Frag Data]]
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* [[Luckau_Protocols:STRUCTURE | STRUCTURE]]
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===Inventory===
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* [[Luckau_Protocols:Inventory | Inventory & Catalog #s]]

Current revision

Tara's Protocols

Tara K. Luckau's Home Page

Conservation Genetics Lab Notebook

Tara's Protocols


Contents

Protocols used in Dr. Rulon Clark's laboratory

Lab Processes


Reagents


Software Programs


Inventory

Personal tools