Biomod/2013/BU/protocols
Boston University
BIOMOD 2013 Design Competition
<html>
<style> body { background: #fff; margin: 0; padding: 0; font-family: 'Open Sans', sans-serif; }
- globalWrapper { background: #ffffff; }
- content { background: none; border-style: none; margin: 0; padding: 0; }
- column-one, #contentSub, .firstHeading, #footer { display: none; }
- BU-header { padding: 15px; width: 100%; background: #000000; height: 20px; }
- BU-title { background: #DF0101; padding: 1.8em 0 1.8em 11.5em; line-height: 1.1em; }
- BU-content { min-width: 768px; width: 1000px; margin: 3.5em auto; }
- BU-body { margin-left: 11em; }
- BU-menu { float: left; padding-top: 0; padding-left: 5px; height: 100%; }
.BU-h1, .BU-h4 { color: white; border-bottom: none; padding: 0; margin-top: 14px; margin-bottom: 14px; }
- BU-header a { color: white; font-size: 19px; }
h1 { font-size: 44px; font-weight: 500; border-bottom: none; } h3 { font-size: 27px; font-weight: 300; border-bottom: none; } h4 { font-size: 23px; font-weight: 300; border-bottom: none; } ul.side-nav { display: block; list-style: none outside none; margin: 0; padding: 0; font-size: 14px; line-height: 1.6;} ul.side-nav li.divider { border-top: 1px solid #2383A1; height: 0; padding: 0; } </style> <link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'> </html>
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 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.