Biomod/2013/BU/protocols
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 250mL Erlenmeyer flask in microwave until boiling begins, stopping to swirl at 15 second intervals.
- Add 500ul of MgCl2 and 5ul 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 50kDa dialysis filter
- Bring total volume is brought to 500μL with isolation buffer.
- Spin sample in centrifuge at 4000 RCF for 15 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.
