Gel Purification of DNA with Macherey-Nagel 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) **Make sure Buffer NT3 has the appropriate volume of ethanol added to it before you go any further.
4) Weigh the gel slice in a colorless tube. Add 2 volumes of buffer Buffer NT to 1 volume of gel (100 mg gel = 200 µL NT). If your gel is > 2% agarose, use 400 µL Buffer NT.
5) 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. Hold the tube up to the light and look for a translucent piece of undissolved gel.
7) Place a Nucleospin Extract II spin column in one of the provided 2 mL collection tubes.
8) To bind DNA, apply the DNA solution to the Nucleospin Extract II spin 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 700 µL of Buffer NT3 (with EtOH previously added) to the column and centrifuge for 1 minute at full speed. Repeat this step with another 700 µL of Buffer NT3.
11) Repeat washing the column with two rounds of 250 µL of Buffer NT3 and centrifuge for 1 minute at full speed. This improves the quality of your final DNA.
12) Discard flow-through first and then spin again for an additional two minues to dry the column.
13) Place the Nucleospin Extract II spin column in a clean, 1.5 mL centrifuge tube.
14) To elute the DNA, add 15 -50 µL of Buffer NE (30 µL is typical) to the CENTER of the membrane. Let this sit for 1 minute at room temperature, then spin full speed for 1 minute. Your DNA is in this small volume. If your DNA is 5-10 kb or larger, preheat buffer NE to 70° C prior to adding to spin column.
15) Reload the eluate onto the same column again and let it sit for one minute. Spin at full speed for one minute and recapture the eluate in the same tube as in step 14. This step increases your yield.
16) Use 1 or 2 µL of this DNA to quantify using the NanDrop. You are ready to do a ligation now.