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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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|
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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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|
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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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|
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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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| #
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