Difference between revisions of "Luckau Protocols"

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==Tara Luckau Protocols==
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{|{{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;" |
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<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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|}
  
Following are the protocols used in Dr. Rulon Clark's laboratory.
 
  
[[NanoDrop]]
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==Protocols used in Dr. Rulon Clark's laboratory==
  
[[Agarose Gel]]
 
  
===NanoDrop===
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===Lab Processes===
====Purpose====
 
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.
 
  
====Protocol====
 
# Assemble cafeteria tray
 
#* Kim Wipes
 
#* 2µL pipette
 
#* pipette tips
 
#* gloves
 
#* USB drive
 
#* water and buffer tubes
 
#* samples
 
# The NanoDrop is located in North Life Sciences, room 325A
 
#*[[Image:NanoDrop1000.jpg]]
 
# Open the NanoDrop software, "ND1000" on the desktop
 
# Choose "Nucleic Acids"
 
# 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
 
# 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
 
# 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 and clicking "Re-Blank" instead of "Blank"
 
# 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 saved files in the same day
 
# Re-initialize the instrument each 30-50 samples, by going back to step 3
 
#* remember to save your data before your re-initialize
 
# Clean up thoroughly! Other people use this instrument!
 
# don't forget to enter data into spreadsheet
 
  
===Agarose Gel===
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* [[Luckau_Protocols:NanoDrop | NanoDrop]]
====Purpose====
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* [[Luckau_Protocols:PCR | PCR]]
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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* [[Luckau_Protocols:Agarose Gel | Agarose Gel]]
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* [[Luckau_Protocols:FragmentAnalysisSubmission | Fragment Analysis Submission]]
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* [[Luckau_Protocols:ShipDryIce | Shipping with Dry Ice]]
  
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]]  [[Image:OwlA2Large_draw.jpg|300 px]]
 
  
====Protocol====
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===Reagents===
# 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:Low TE | Low TE]]
|-
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* [[Luckau_Protocols:Tris-Cl | Tris-Cl]]
! 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:KCl | KCl]]
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* [[Luckau_Protocols:MgCl2 | MgCl<sub>2</sub>]]
| small || 50 || _ || 1 || 5 || 4-6 || 80
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* [[Luckau_Protocols:TAE | TAE]]
|-
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* [[Luckau_Protocols:1Kb Ladder | 1Kb Ladder]]
| 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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* [[Luckau_Protocols:dNTPs | dNTPs]]
|-
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* [[Luckau_Protocols:PCR Buffers A-H | PCR Buffers A-H]]
| 36-tooth comb: 3-5
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* [[Luckau_Protocols:PrimerResuspension | Primer Resuspension]]
|-
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| large || _ || _ || _ || _ || _ || 170
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|}
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===Software Programs===
#
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* [[Luckau_Protocols:STRUCTURE | STRUCTURE]]
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===Inventory===
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* [[Luckau_Protocols:Inventory | Inventory & Catalog #s]]

Revision as of 12:37, 12 March 2013

Tara's Protocols

Tara K. Luckau's Home Page

Conservation Genetics Lab Notebook

Tara's Protocols


Protocols used in Dr. Rulon Clark's laboratory

Lab Processes


Reagents


Software Programs


Inventory