Skatebro:Electrophoresis Gels

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(Tips)
Current revision (05:10, 9 February 2008) (view source)
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[[Skatebro:Project08]]
 
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===Tips===
 
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*Heat flasks before adding liquid gel to prevent solidification
 
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*Be really careful with EB (1 or 2 microL)
 
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*Run 50 mL small gels at 85 volts with amp not restrictive
 
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*Use an appropriate ladder
 
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*Record the order of your samples on a post-it note
 
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*Position gel in imager using upper white and then close door for transilluminator
 
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*Left hand=dirty, right hand=clean
 
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==Steps==
 
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#Prepare a gel holder with comb (use fat ends bc make bigger wells)
 
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#Start thawing loading dye and ladder
 
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#Get a small flask, heat, add ~40mL 1% agarose from the ~70C incubator
 
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#Put on gloves and take flask to the gel area
 
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#Carefully add 2μL of Ethidium Bromide to the agarose and swirl gently
 
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#Pour gel - avoid bubbles/clumps
 
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#Wait 20 minutes for the gel to solidify
 
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#While waiting, prepare samples to load:
 
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#*Change gloves
 
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#*Cut a piece of parafilm
 
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#*Make dots of loading dye in a row on the paraflim.
 
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#**As you need to run at least 100ng of DNA in each well to see bands, check your [DNA] and If you're using 6x loading dye, add 5 parts sample to 1 part loading dye, and if using 2x loading dye, add 1 part sample to 1 part loading dye. (generally works out to be about 5μL of loading dye + 5μL of sample if use the 2x loading dye, and 1μL of loading dye + 5μL of sample if use the 6x dye)
 
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#*Add dna samples to loading dye dots. MIx by carefully pipetting up and down.
 
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#**Make sure to add ladder to the first dot.
 
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#**'''Record the order of your samples on a post-it note'''
 
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#Place hardened gel in a gel box with the wells on the left side. Add enough 1x TAE buffer to cover the gel.
 
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#Load the samples without puncturing gel
 
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#Run 50 mL small gels at 85 volts with amp not restrictive for ~45 minutes (until blue band ~3/4 the way down gel)
 
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#'''wearing gloves''': remove gel and place on a paper towel.
 
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#change your right glove...now this is your clean hand, to operate the computer with
 
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#using your "dirty" left hand, set the machine to "upper white" and center gel
 
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#close the door and set light to "transilluminator"
 
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#*Hit the green button to take an image.  When an image pops up, hit the red button to capture the image and turn off the light.  If it's not bright enough, you can increase the exposure time.
 
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#*Go to File->Export Image and save the image as a .jpg. Also print a copy.
 
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#Put the gel into the biohazard waste container, and rinse off the plastic gel holder.
 
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#Clean up any spills/mess
 

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