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If you are purifying DNA that is already in aqueous solution, then proceed directly to step 3. If the DNA is being gel purified, then begin with step 1.
1. Excise DNA band from ethidium bromide-stained gel using a fresh razor blade and place into a clean Eppendorf tube. Use long-wave UV light for as short a time as possible to visualize the DNA.
2. Determine the approximate volume of the gel slice by weight. If the slice weighs more than 0.4 g, it may be necessary to divide it into two fragments and continue the procedure in two Eppendorf tubes.
3. Add 3 volumes of NaI solution (BIO 101).
If DNA is not contained in agarose, go directly to step 5.
4. If agarose is present, place the tube in a 50 degree water bath. After a minute, mix the contents of the tube and return it to the water bath. After approximately 5 minutes, the agarose should be completely dissolved. Do not proceed until the agarose is completely dissolved. Do not leave the tube in the water bath for any longer than is necessary to dissolve the agarose.
5. Vortex the Glassmilk tube for 1 full minute, holding the tube horizontally.
6. Add 5 ul of Glassmilk suspension to the solution and mix by inverting rapidly. If there is more than 5 ug of DNA present, then 10 ul of Glassmilk suspension should be used.
7. Place tube in the Labquake rotator and let rotate for 5 minutes at room temperature. The DNA binds to the silica matrix during this step.
8. Pellet the silica matrix with bound DNA by spinning in a microcentrifuge for 5 seconds at full speed (Wait for the centrifuge to come to full speed, count to five, and let it come to a halt).
9. Carefully aspirate off the supernatant and resuspend the pellet in 500 ul of NEW WASH (BIO 101) by pipetting up and down.
10. Spin for 5 seconds in a microcentrifuge and remove the supernatant. Repeat this wash procedure two more times. These washes remove the proteins and most of the RNA because they do not bind to the silica matrix, as well as removing any salts or ethidium bromide.
11. Spin the tube in a Speed-Vac concentrator to remove the last traces of NEW WASH.
12. Resuspend pellet in 25 ul of DD-H2O (or any other low salt buffer such as TE) by pipetting up and down. Incubate the tube in a 50 degree water bath for 3 minutes.\ The DNA does not bind to the silica matrix in the absence of salts and is eluted during this step.
13. Spin for 30 seconds in a microcentrifuge to make a solid pellet. Carefully remove 20 ul of the supernatant containing the cleaned DNA and place in a fresh Eppendorf tube.
If a small amount of Glassmilk is carried over, it does not interfere with subsequent use of the DNA but can be removed by briefly spinning the tube before withdrawing any of the solution.
14. Resuspend the pellet in an additional 20 ul of your low-salt buffer, incubate, spin, and transfer 20 ul of the supernatant into the tube containing the clean DNA.
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