Difference between revisions of "Biomod/2013/BU/protocols"

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<h3>Protocols</h3>
 
<h3>Protocols</h3>
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<h4>Gel Electrophoresis</h4>
 
<h4>Gel Electrophoresis</h4>
 +
---------------------------
 
<OL type="I">
 
<OL type="I">
 
<LI><h5>Gel Prep:</h5>
 
<LI><h5>Gel Prep:</h5>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5mL 5X Agarose Gel Buffer<br />
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5mL 5X Agarose Gel Buffer<br />
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;45mL ddH<sub>2</sub>O<br />
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;45mL ddH<sub>2</sub>O<br />
<LI> Gently mix and heat in 250mL Erlenmeyer flask in microwave until boiling begins, stopping to swirl at 15 second intervals.  
+
<LI> Gently mix and heat in a 250mL Erlenmeyer flask in microwave until boiling begins, stopping to swirl at 15 second intervals.  
<LI>Add 500ul of MgCl<sub>2</sub> and 5ul SybrSafe gel dye.  
+
<LI>Add 500μL of MgCl<sub>2</sub> and 5μL SybrSafe gel dye.  
 
<LI>Gently swirl the flask to mix contents
 
<LI>Gently swirl the flask to mix contents
 
<LI>Pour the contents into a gel mold.  
 
<LI>Pour the contents into a gel mold.  
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<h4>TEM Prep</h4>
 
<h4>TEM Prep</h4>
 +
-------------------
 
<UL>
 
<UL>
 
<LI>Select a TEM grid using specialized forceps and set the forceps with the grid down on a flat surface.  
 
<LI>Select a TEM grid using specialized forceps and set the forceps with the grid down on a flat surface.  
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<h4>Measuring Concentrations via NanoDrop</h4>
 
<h4>Measuring Concentrations via NanoDrop</h4>
 +
---------------------------------------------
 
<UL>
 
<UL>
 
<LI>Open the NanoDrop program and select Nucleic Acids.  
 
<LI>Open the NanoDrop program and select Nucleic Acids.  
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<h4>Purification via Dialysis Filters</h4>
 
<h4>Purification via Dialysis Filters</h4>
 +
------------------------------------
 
<UL>
 
<UL>
<LI>Pipette entire sample volume into the 500uL 50kDa dialysis filter
+
<LI>Pipette entire sample volume into the 500uL 30kDa dialysis filter
<LI>Bring total volume is brought to 500μL with isolation buffer.  
+
<LI>Bring total volume is brought to 500μL withTE buffer.  
<LI>Spin sample in centrifuge at 4000 RCF for 15 minutes.  
+
<LI>Spin sample in centrifuge at 14000 RCF for 5 minutes.  
 
<LI>Discard staples and the refill filter to total volume of 500uL.
 
<LI>Discard staples and the refill filter to total volume of 500uL.
 
<LI>Repeat for a maximum of 3 spins with a single filter.  
 
<LI>Repeat for a maximum of 3 spins with a single filter.  
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<h4>Gel Purification</h4>
 
<h4>Gel Purification</h4>
 +
------------------------
 
<UL>
 
<UL>
 
<LI>Run the samples in a 1.5% agarose gel until product band and misfolded band separate sufficiently.  
 
<LI>Run the samples in a 1.5% agarose gel until product band and misfolded band separate sufficiently.  

Revision as of 07:15, 26 July 2013

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Protocols


Gel Electrophoresis


  1. Gel Prep:
    • Make the gel mixture:
           (For a 1% Agarose gel)
           0.5g Agarose powder
           5mL 5X Agarose Gel Buffer
           45mL ddH2O
    • Gently mix and heat in a 250mL Erlenmeyer flask in microwave until boiling begins, stopping to swirl at 15 second intervals.
    • Add 500μL of MgCl2 and 5μL SybrSafe gel dye.
    • Gently swirl the flask to mix contents
    • Pour the contents into a gel mold.
    • Place the well comb in the mold and freeze for 20 minutes.
  2. Sample Prep:

    Aliquot the desired volume of sample to be run in the gel, ensuring not to exceed the well capacity. Add loading dye and mix via pipetting.

  3. Running the Gel
    • Place the gel in the electrophoresis chamber
    • Fill the chamber with 300mL 0.5X Gel Buffer.
    • Remove the comb and load the sample into the wells.
    • Connect the electrodes and run the gel at the desired voltage for the desired length of time.
    • To prevent overheating of the gel, surround the gel electrophoresis chamber with ice.

TEM Prep


  • Select a TEM grid using specialized forceps and set the forceps with the grid down on a flat surface.
  • Pipette 3uL of the desired sample onto the grid and let it sit for 2 minutes.
  • Wick away any excess moisture with filter paper.
  • Dip the TEM grid, sample side down, into a drop of uranyl acetate and hold for 30 seconds.
  • Wick away any excess moisture with filter paper.
  • Dip the TEM grid, sample side down, into a drop of ddH2O and hold for 30 seconds.
  • Wick away any excess moisture.
  • Let the grid sit in the forceps on a flat surface for 1 minute until the surface of the grid is completely dry.

Measuring Concentrations via NanoDrop


  • Open the NanoDrop program and select Nucleic Acids.
  • Blank the instrument with 2μL of the solution which the sample is suspended in.
  • Wipe away any excess solution with a Kimwipe
  • Pipette 2μL of the sample onto the instrument and click "Measure."
  • Repeat for as many samples as necessary.
  • Rinse the instrument with 2μL ddH2O in between samples.

Purification via Dialysis Filters


  • Pipette entire sample volume into the 500uL 30kDa dialysis filter
  • Bring total volume is brought to 500μL withTE buffer.
  • Spin sample in centrifuge at 14000 RCF for 5 minutes.
  • Discard staples and the refill filter to total volume of 500uL.
  • Repeat for a maximum of 3 spins with a single filter.
  • To retrieve the sample from the filter, the filter column is inverted and placed in a new tube. This tube is then spun at 2000 RF for 2 minutes.

Gel Purification


  • Run the samples in a 1.5% agarose gel until product band and misfolded band separate sufficiently.
  • Cut the product band from the gel, leaving behind as much excess gel as possible.
  • Transfer the gel band to a gel filtration tube and freeze the sample for 10 minutes.
  • Spin the sample down for 14 minutes at 5000 RCF.
  • Pipette out the recovered sample.