Biomod/2013/BU/protocols: Difference between revisions
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<h3>Protocols</h3> | <h3>Protocols</h3> | ||
<h4>Gel Electrophoresis</h4> | <h4>Gel Electrophoresis</h4> | ||
--------------------------- | |||
< | <OL type="I"> | ||
<LI><h5>Gel Prep:</h5> | |||
<UL> | <UL> | ||
<LI>Make the gel mixture:<br /> | <LI>Make the gel mixture:<br /> | ||
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5mL 5X Agarose Gel Buffer<br /> | 5mL 5X Agarose Gel Buffer<br /> | ||
45mL ddH<sub>2</sub>O<br /> | 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 | <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> | ||
< | <LI><h5>Sample Prep:</h5> | ||
< | |||
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 /> | ||
< | <LI><h5>Running the Gel</h5> | ||
< | |||
<UL> | <UL> | ||
<LI>Place the gel in the electrophoresis chamber | <LI>Place the gel in the electrophoresis chamber | ||
Line 36: | Line 31: | ||
<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> | <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. | ||
Line 50: | Line 47: | ||
<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. | ||
Line 60: | Line 58: | ||
<h4>Purification via Dialysis Filters</h4> | <h4>Purification via Dialysis Filters</h4> | ||
------------------------------------ | |||
<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
Boston University
BIOMOD 2013 Design Competition
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Protocols
Gel Electrophoresis
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.
- Make the gel mixture:
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.
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.