Difference between revisions of "Team:NYU Shanghai/Protocols"

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       <li>Carefully transfer the spin column into a sterile 1.5 mL tube and add 50-100 µL (> 50 µL) Sterile ddH20 or Elution Buffer into the center of the column and let it stand for 2 minutes. Elute the DNA by centrifugation at 13,000 rpm for 1 minute. Reload the eluate into the column and elute again.</li>
 
       <li>Carefully transfer the spin column into a sterile 1.5 mL tube and add 50-100 µL (> 50 µL) Sterile ddH20 or Elution Buffer into the center of the column and let it stand for 2 minutes. Elute the DNA by centrifugation at 13,000 rpm for 1 minute. Reload the eluate into the column and elute again.</li>
 
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     This is how you do gel extraction.
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Estimated time:
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  <br><br>Materials
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  <br>MinElute Midi Gel Extraction Kit
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  <br><br>Procedure
 +
     <ol>
 +
    <li>Pre-weigh a 2.0mL microcentrifuge tube</li>
 +
    <li>Excise DNA fragment from agarose gel with clean, sharp scalpel. Place in the pre-weighed microcentrifuge tube. Minimize the exposure of the DNA samples under UV and amount of gel cut with DNA.</li>
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    <li>Weigh the tube and calculate the weight of the gel. For every 1 volume of gel (100mg = 100µl) add 3 times the volume of Buffer QG. The maximum amount of gel slice per spin column is 400mg.For >2% agarose gels, add 6 volumes of Buffer QG.</li>
 +
    <li>Incubate at 50ºC for 10 minutes (or until the gel has completely dissolved). Vortex the tube every 2-3 minutes during incubation to help dissolve the gel. If color is orange or violet after gel slice has dissolved, add 10µl 3M sodium acetate, pH 5.0. The color of the mixture will turn to yellow. </li>
 +
    <li>Add 1 gel volume of isopropanol to the sample and mix by inverting.</li>
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    <li>Place a MinElute spin column in a provided 2mL collection tube. Apply the sample in the 2.0mL microcentrifuge tube to the MinElute column and centrifuge for 1 min at 13,000 rpm. Discard the flow-through and place the MinElute column back into the same collection tube. If the sample volume exceeds 800µl, simply reload and spin again.</li>
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    <li>Add 500µl Buffer QG to the MinElute column and centrifuge for 1 minute at 13,000 rpm. Discard flow-through and place the MinElute column back into the same collection tube.</li>
 +
    <li>Add 750µl Buffer PE to MinElute column and centrifuge for 1 minute at 13,000 rpm. Discard flow-through and place the MinElute column back into the same collection tube. If the DNA will be used for salt-sensitive applications, such as direct sequencing and blunt-ended ligation, let the column stand 2-5 minute after addition of Buffer PE.</li>
 +
    <li>Centrifuge the column in the 2mL collection tube for 1 minute at 13,000 rpm again to remove residual ethanol.</li>
 +
    <li>Place each MinElute column into a clean 1.5mL microcentrifuge tube. To elute DNA, add 10µl Buffer EB or water to the center of the MinElute membrane. Let the column stand for 1 minute, and centrifuge the column for 1 minute at 13,000rpm.</li>
 +
    <li>Repeat step 12 but this time load the flow-through back to the membrane. Let the column stand for 1 minute, and centrifuge the column for 1 minute at 13,000rpm.</li>
 +
    <li>Check Gel extraction product with Nanodrop</li>
 
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Revision as of 02:12, 15 September 2015

Protocols

Recipes

Making Color

3A Assembly