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<h3>Protocols</h3>
<h3>Protocols</h3>
--------------------
 


<h4>Gel Electrophoresis</h4>
<h4>Gel Electrophoresis</h4>
 
---------------------------
<b>Gel Prep</b>:
<OL type="I">
 
<LI><h5>Gel Prep:</h5>
For 1% Agarose Gel: <br />
<UL>
<UL>
<LI>0.5g Agarose powder
<LI>Make the gel mixture:<br />
<LI>5mL 5X Agarose Gel Buffer
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(For a 1% Agarose gel)<br />
<LI>45mL ddH<sub>2</sub>O
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0.5g Agarose powder<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5mL 5X Agarose Gel Buffer<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;45mL ddH<sub>2</sub>O<br />
<LI> Gently mix and heat in a 250mL Erlenmeyer flask in microwave until boiling begins, stopping to swirl at 15 second intervals.
<LI>Add 500μL of MgCl<sub>2</sub> and 5μL SybrSafe gel dye.
<LI>Gently swirl the flask to mix contents
<LI>Pour the contents into a gel mold.
<LI>Place the well comb in the mold and freeze for 20 minutes.
</UL>
</UL>
 
<LI><h5>Sample Prep:</h5>
 
Add 500ul of MgCl<sub>2</sub> and 5ul SybrSafe gel dye. Gently swirl the flask to mix contents and then pour the contents into a gel mold. Place the well comb in the mold and freeze for 20 minutes.
 
<b>Gel Buffer</b>
 
To prepare the buffer that fills the gel electrophoresis chamber, pour 30mL of the 5X  buffer into a 500mL flask and dilute the total volume of the solution to 300mL. Set aside until the gel is solidified.
 
<b>Sample Prep</b>
 
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.
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.
 
<br />
<b>Running the Gel</b>
<LI><h5>Running the Gel</h5>
 
<UL>
Place the gel in the electrophoresis chamber and fill the chamber with the 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.  
<LI>Place the gel in the electrophoresis chamber
 
<LI>Fill the chamber with 300mL 0.5X Gel Buffer.  
To prevent overheating of the gel, surround the gel electrophoresis chamber with ice.  
<LI>Remove the comb and load the sample into the wells.  
 
<LI>Connect the electrodes and run the gel at the desired voltage for the desired length of time.  
<LI>To prevent overheating of the gel, surround the gel electrophoresis chamber with ice.  
</UL>
</OL>


<h4>TEM Prep</h4>
<h4>TEM Prep</h4>
 
-------------------
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. Afterwards, wick away any excess moisture with filter paper. Next dip the TEM grid, sample side down, into a drop of uranyl acetate and hold for 30 seconds. Afterwards, wick away any excess moisture with filter paper. Lastly, dip the TEM grid, sample side down, into a drop of ddH<sub>2</sub>O and hold for 30 seconds. Afterwards 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.  
<UL>
 
<LI>Select a TEM grid using specialized forceps and set the forceps with the grid down on a flat surface.  
<LI>Pipette 3uL of the desired sample onto the grid and let it sit for 2 minutes.  
<LI>Wick away any excess moisture with filter paper.  
<LI>Dip the TEM grid, sample side down, into a drop of uranyl acetate and hold for 30 seconds.  
<LI>Wick away any excess moisture with filter paper.  
<LI>Dip the TEM grid, sample side down, into a drop of ddH<sub>2</sub>O and hold for 30 seconds.  
<LI>Wick away any excess moisture.  
<LI>Let the grid sit in the forceps on a flat surface for 1 minute until the surface of the grid is completely dry.  
</UL>


<h4>Measuring Concentrations via NanoDrop</h4>
<h4>Measuring Concentrations via NanoDrop</h4>
 
---------------------------------------------
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 and then pipette 2μL of the sample onto the instrument and click "Measure." Repeat for as many samples as necessary. In between samples, rinse the instrument with 2μL ddH<sub>2</sub>O.  
<UL>
 
<LI>Open the NanoDrop program and select Nucleic Acids.  
<LI>Blank the instrument with 2μL of the solution which the sample is suspended in.  
<LI>Wipe away any excess solution with a Kimwipe  
<LI>Pipette 2μL of the sample onto the instrument and click "Measure."  
<LI>Repeat for as many samples as necessary.  
<LI>Rinse the instrument with 2μL ddH<sub>2</sub>O in between samples.
</UL>


<h4>Purification via Dialysis Filters</h4>
<h4>Purification via Dialysis Filters</h4>
 
------------------------------------
During small scale experiments, 500μL 50kDa dialysis filters were used. The entire sample volume is pipetted into the filter and then the total volume is brought to 500μL with isolation buffer. The sample is spun in a centrifuge at 4000 RCF for 15 minutes. The staples are discarded and the volume of the filter is refilled to 500μL. The sample is spun again. This process is repeated 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.
<UL>
<LI>Pipette entire sample volume into the 500uL 30kDa dialysis filter
<LI>Bring total volume is brought to 500μL withTE buffer.  
<LI>Spin sample in centrifuge at 14000 RCF for 5 minutes.  
<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>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.
</UL>


<h4>Gel Purification</h4>
<h4>Gel Purification</h4>
 
------------------------
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.
<UL>
<LI>Run the samples in a 1.5% agarose gel until product band and misfolded band separate sufficiently.  
<LI>Cut the product band from the gel, leaving behind as much excess gel as possible.  
<LI>Transfer the gel band to a gel filtration tube and freeze the sample for 10 minutes.  
<LI>Spin the sample down for 14 minutes at 5000 RCF.  
<LI>Pipette out the recovered sample.
</UL>

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.
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