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<h3>Protocols</h3>
<p style="font-size:30px">Protocols</p>
--------------------


<h4>Gel Electrophoresis</h4>
<p style="font-size:20px">Gel Electrophoresis</p>
 
---------------------------
<b>Gel Prep</b>
<OL type="I">
<br />
<LI><p style="font-size:15px">Gel Prep</p>
<UL>
<UL>
<LI>Make the gel mixture:<br />
<LI>Make the gel mixture:<br />
Line 14: Line 13:
&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 a 250mL Erlenmeyer flask in microwave until boiling begins, stopping to swirl at 15 second intervals.  
<LI> Gently mix and heat in 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>Add 500ul of MgCl<sub>2</sub> and 5ul 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.  
<LI>Place the well comb in the mold and freeze for 20 minutes.
<LI>Place the well comb in the mold and freeze for 20 minutes.
</UL>
</UL>
<br />
<LI><p style="font-size:15px">Sample Prep</p>
<br />
<b>Sample Prep</b>
<br />
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 />
<br />
<br />
<LI><p style="font-size:15px">Running the Sample</p>
<b>Running the Gel</b>
<br />
<UL>
<UL>
<LI>Place the gel in the electrophoresis chamber
<LI>Place the gel in the electrophoresis chamber
Line 37: Line 30:
<LI>To prevent overheating of the gel, surround the gel electrophoresis chamber with ice.  
<LI>To prevent overheating of the gel, surround the gel electrophoresis chamber with ice.  
</UL>
</UL>
</OL>


<h4>TEM Prep</h4>
<p style="font-size:20px">TEM Prep</p>
-------------------
<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.  
Line 50: Line 45:
</UL>
</UL>


<h4>Measuring Concentrations via NanoDrop</h4>
<p style="font-size:20px">Measuring Concentrations via Nanodrop</p>
---------------------------------------------
<UL>
<UL>
<LI>Open the NanoDrop program and select Nucleic Acids.  
<LI>Open the NanoDrop program and select Nucleic Acids.  
Line 60: Line 56:
</UL>
</UL>


<h4>Purification via Dialysis Filters</h4>
<p style="font-size:20px">Purification via dialysis filters</p>
------------------------------------
<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.  
Line 70: Line 67:
</UL>
</UL>


<h5>Gel Purification</h5>
<p style="font-size:20px">Gel Purification</p>
------------------------
<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.  
Line 77: Line 75:
<LI>Spin the sample down for 14 minutes at 5000 RCF.  
<LI>Spin the sample down for 14 minutes at 5000 RCF.  
<LI>Pipette out the recovered sample.
<LI>Pipette out the recovered sample.
</UL>
<p style="font-size:20px">Polymer Synthesis</p>
------------------------------------------
<UL>
<LI>React hetero-bifunctionalized (NHS ester & maleimide) 10 kDa polyethylene glycol with amine functionalized oligonucleotide in pH 8 PBS at a ratio of 1:2.4 (PEG:oligo). 
<LI>Vortex for 1.5 hr
<LI>Using a 3 kDa dialysis centrifuge tube spin 3 time at 14,000 rcf for 30 min against D<sub>2</sub>O
<LI>Analyze by <sup>1</sup>H NMR spectroscopy to verify the disappearance of the activated ester singlet peak
<LI>Lyophilize
<LI>React oligo-functionalized PEG with cysteine-containing peptide in DMSO/pH 7 PBS (1:3) at a ratio of 1:2.2 (PEG:peptide)
<LI>Vortex overnight
<LI>Dialyze against water for two days using a 6kDa cutoff
<LI>Analyze by <sup>1</sup>H NMR spectroscopy to verify the disappearance of the maleimide singlet peak
<LI>Lyophilize
</UL>
<p style="font-size:20px">Cell Assay</p>
------------------------------------------
<UL>
<LI>Apply sample to glass coverslip containing primary hippocampal neurons obtained from rats
<LI>Let sit for one hour; add buffer to prevent sample from drying out
<LI>Rinse slide repeatedly with buffer to remove all sample
<LI>Image immediately using brightfield and fluorescence microscopy
</UL>
<p style="font-size:20px">in vivo Study</p>
------------------------------------------
<UL>
<LI>Anesthetize mouse and mount on surgery table
<LI>Open the scalp and level the head using a stereotaxic instrument
<LI>Load at least 6uL sample into a syringe and load syringe into syringe pump
<LI>Drill a hole in the skull above the desired brain coordinates
<LI>Lower the needle tip to the first injection site
<LI>Inject 3uL over 10 minutes
<LI>Wait 10 minutes
<LI>Remove needle and wait 30 minutes
<LI>Euthanize mouse by cervical dislocation followed immediately by decapitation
<LI>Using scissors, remove the brain from the skull and flash freeze in a dry ice/methylbutane solution
<LI>Embed the brain in OCT embedding solution
<LI>Slice the brain at 30μM on a cryostat
<LI>Mount slices immediately onto a coverslip
<LI>Image samples using brightfield and fluorescence microscopy
</UL>
</UL>

Latest revision as of 17:46, 26 October 2013

Boston University

BIOMOD 2013 Design Competition

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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 Sample

    • 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.

Polymer Synthesis

  • React hetero-bifunctionalized (NHS ester & maleimide) 10 kDa polyethylene glycol with amine functionalized oligonucleotide in pH 8 PBS at a ratio of 1:2.4 (PEG:oligo).
  • Vortex for 1.5 hr
  • Using a 3 kDa dialysis centrifuge tube spin 3 time at 14,000 rcf for 30 min against D2O
  • Analyze by 1H NMR spectroscopy to verify the disappearance of the activated ester singlet peak
  • Lyophilize
  • React oligo-functionalized PEG with cysteine-containing peptide in DMSO/pH 7 PBS (1:3) at a ratio of 1:2.2 (PEG:peptide)
  • Vortex overnight
  • Dialyze against water for two days using a 6kDa cutoff
  • Analyze by 1H NMR spectroscopy to verify the disappearance of the maleimide singlet peak
  • Lyophilize

Cell Assay

  • Apply sample to glass coverslip containing primary hippocampal neurons obtained from rats
  • Let sit for one hour; add buffer to prevent sample from drying out
  • Rinse slide repeatedly with buffer to remove all sample
  • Image immediately using brightfield and fluorescence microscopy

in vivo Study

  • Anesthetize mouse and mount on surgery table
  • Open the scalp and level the head using a stereotaxic instrument
  • Load at least 6uL sample into a syringe and load syringe into syringe pump
  • Drill a hole in the skull above the desired brain coordinates
  • Lower the needle tip to the first injection site
  • Inject 3uL over 10 minutes
  • Wait 10 minutes
  • Remove needle and wait 30 minutes
  • Euthanize mouse by cervical dislocation followed immediately by decapitation
  • Using scissors, remove the brain from the skull and flash freeze in a dry ice/methylbutane solution
  • Embed the brain in OCT embedding solution
  • Slice the brain at 30μM on a cryostat
  • Mount slices immediately onto a coverslip
  • Image samples using brightfield and fluorescence microscopy
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