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| ==Tara Luckau Protocols== | | {|{{table}} width="900" |
| 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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| | |align="right" style="background-color: #9DB68C;" | |
| | <span style="font-size:16px;"> [[User:Tara_K._Luckau | Tara K. Luckau's Home Page]] </span> |
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| | [[User:Tara_K._Luckau/Notebook/Team_ConGen | Conservation Genetics Lab Notebook]] |
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| | [[Luckau_Protocols | Tara's Protocols]] |
| | |} |
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| | ==Protocols used in Dr. Rulon Clark's laboratory== |
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| | ===Lab Processes=== |
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| | * [[Luckau_Protocols:NanoDrop | NanoDrop]] |
| | * [[Luckau_Protocols:PCR | PCR]] |
| | * [[Luckau_Protocols:Agarose Gel | Agarose Gel]] |
| | * [[Luckau_Protocols:FragmentAnalysisSubmission | Fragment Analysis Submission]] |
| | * [[Luckau_Protocols:ShipDryIce | Shipping with Dry Ice]] |
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| | ===Reagents=== |
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| | * [[Luckau_Protocols:Low TE | Low TE]] |
| | * [[Luckau_Protocols:Tris-Cl | Tris-Cl]] |
| | * [[Luckau_Protocols:KCl | KCl]] |
| | * [[Luckau_Protocols:MgCl2 | MgCl<sub>2</sub>]] |
| | * [[Luckau_Protocols:TAE | TAE]] |
| | * [[Luckau_Protocols:1Kb Ladder | 1Kb Ladder]] |
| | * [[Luckau_Protocols:dNTPs | dNTPs]] |
| | * [[Luckau_Protocols:PCR Buffers A-H | PCR Buffers A-H]] |
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| | ===Software Programs=== |
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| ===NanoDrop===
| | * [[Luckau_Protocols:STRUCTURE | STRUCTURE]] |
| ====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=== | | ===Inventory=== |
| ====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====
| | * [[Luckau_Protocols:Inventory | Inventory & Catalog #s]] |
| # 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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| | small || 50 || _ || 1 || 5 || 4-6 || 80
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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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| | 36-tooth comb: 3-5
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| | large || _ || _ || _ || _ || _ || 170
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| |} | |
| # | |
