Gel Purification of DNA with Qiaquick Kit
1) Run the fragment(s) on a gel and photograph the gel. Remember to use a MW marker. Also, use as low a percentage gel as you can to resolve your bands.
2) Cut out the band(s) of interest using a razor blade and the UV box with the hinged plexiglass covering. Cut as close to the band as possible to minimize the volume of the gel piece. Use as low a level UV light for as little time as possible. Protect your eyes and skin from the UV.
3) Weigh the gel slice in a colorless tube. Add 3 volumes of buffer QG to 1 volume of gel (100 mg = 100 µL).
4) Incubate in 50° C waterbath for 10 minutes, or until the gel slice has COMPLETELY dissolved. You can vortex every 2-3 minutes to speed up the dissolving process.
5) After the gel is completely dissolved, check that the color of the solution is yellow.
6) If the fragment is less than 500 bp or bigger than 4kb, add 1 gel volume of isopropanol to the solution and vortex for 30 seconds. Otherwise, skip to the next step.
7) Place a QIAquick spin column in one of the provided 2 mL collection tubes.
8) To bind DNA, apply the DNA solution to the QIAquick column and centrifuge as full speed for one minute. The maxiumum volume you can load at a time is 800 µL. If your volume is larger, then reload the same column and spin a second time.
9) Discard flow-through from step 8 and place the column back in the same collection tube.
10) To wash, add 0.75 mL of buffer PE to the column and centrifuge for 1 minute at full speed.
11) Discard flow-through first and then spin again for an additional one minute.
12) Place the QIAquick column in a clean, 1.5 mL centrifuge tube.
13) To elute the DNA, add 30 µL of buffer EB to the CENTER of the QIAquick membrane. Let this sit for 1 minute, then spin full speed for 1 minute. Your DNA is in this small volume.
14) Use 1 or 2 µL of this DNA to quantify using the NanDrop. You are ready to do a ligation now.
© Copyright 2007 Department of Biology, Davidson College, Davidson, NC 28036
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