Gene Clean

GeneClean Protocol
1. Run your samples on a 1% GTG agarose gel. Take a picture of the gel. Mark the bands to be cut out.
2. Use razor to cut the bands out of the gel. Place the bands in a 1.7 ml centrifuge tube that was weighed before addition of the band.
Weigh the tube + band. Record both. Determine the weight of the band.
3. Add 3x NaI (meaning 3 x weight of the band of agarose). For smaller DNA, 200-500 bp, add 1/10 volume of TBE Modifier to the NaI
solution to lower pH of the NaI/Glassmilk/DNA mixture to  6.5 to maximize smaller DNA binding efficiencies.
4. Incubate the solution for 10 minutes at 45-55C. Mix after 2 minutes.
5. After vortexing the glassmilk for about 1 minute, add 5 l to the tubes.
6. Mix well and incubate the solution for 15 minutes at room temperature. Mix every 5 minutes to keep the glassmilk suspended.
7. Centrifuge at high speed for 5 seconds. Pour the supernate into another centrifuge tube. (Keep it just in case all the DNA did not bind.)
8. Resuspend the pellet in 500 l of New Wash solution.
9. Centrifuge at high speed. Remove the supernate by aspiration.
10. Repeat steps 8 and 9 twice more for a total of three washes.
11. Recentrifuge to remove residual ethanol.
12. Resuspend the pellet in 20 l of dH2O.
13. Centrifuge at high speed for 2 minutes. Transfer 15 µl of the supernate to another tube.
14. Repeat steps 12 and 13. Combine the supernates from steps 13 and 14 into one tube for a total of 30l.
15. Run an analytical gel to determine if the correct band was cut or if the DNA was lost.