Team:Wash U/Protocol
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=='''Gel Extraction'''== | =='''Gel Extraction'''== | ||
<font size="2"> | <font size="2"> | ||
- | : | + | :Gel Extraction is intended to recover and reuse DNA that has been purified by gel electrophoresis. The process re-isolates DNA and removes all primers, dyes, and ethidium bromide that may still by present in the band of DNA in the gel, leaving up to 95% of the original DNA. We will be using the GenElute Gel Extraction Kit from Sigma-Aldrich so individual formulas and solutions may be unattainable. Please contact them for more details. |
'''Materials''' | '''Materials''' | ||
- | * | + | * gel with desired DNA band |
+ | * Column preparation solution | ||
+ | * Gel Solubilization Solution | ||
+ | * Wash Solution Concentrate G | ||
+ | * Elution Solution | ||
+ | * GenElute Binding Column G | ||
+ | * spin columns | ||
+ | * Ethanol | ||
+ | * Isopropanol | ||
+ | * 3M Sodium Acetate Buffer (pH 5.2) | ||
'''Procedures''' | '''Procedures''' | ||
- | # | + | # Dilute the Wash Solution Concentrate G with 48mL of ethanol. |
+ | # Cut out the desired band of DNA from the gel leaving as little excess gel as possible. | ||
+ | # Weight the excised gel and place into a microcentrifuge tube. Add 3 gel volumes of Gel Solubilization Solution to the tube (for every 100mg of gel, add 300uL of solution). Incubate tube at 60C for 10 minutes or until gel is completely dissolved. Vortex occasionally to ensure gel is evenly dissolved. | ||
+ | # Prepare the binding column by adding 500uL of Column Preparation Solution to the column and centrifuging for 1 minute. Discard the flow through. | ||
+ | # Check the color of the tube containing the dissolved gel. If the solution is red, add 10uL of 3M Sodium Acetate to the mix until the solution turns yellow. | ||
+ | # Add 1 gel volume of 100% isopropanol and mix (may need slightly more isopropanol for gels greater than 2%). | ||
+ | # Load the gel solution into the binding column and centrifuge for 1 minute and discard the flow through. | ||
+ | # Add 700uL of Wash Solution to the column and cetrifuge for 1 minute and discard the flow through. Centrifuge the column again for 1 minute to remove excess ethanol. | ||
+ | # Transfer the column to a new collection tube. Add 50uL of preheated Elution solution (65C) to the column and centrifuge for 1 minute. The desired DNA is now in the collection tube. | ||
Revision as of 16:27, 15 July 2009