DNA extraction from gel - (Apr/18/2007 )
In case your lab doesn't want to spend money for a kit, there is a very oldfashioned method using phenol:
work under a hood or a good ventilaed place
1. Cut the band with your product and put it into a tube (you can freeze it at -20°C to make a break or go home to have a life)
2. Add about the same volume of phenol. Use a 1000µl pipette to crush the agarose block as good as possible. The smaller the pieces the better.
3. Vortex like a maniac (3 to 5 minutes)
4. Centrifuge for 10 minutes at 13.000 rpm
5. You should see two phases. The upper one is the aquatious phase and should contain the DNA. Use a pipette to carefully transfer it into a new tube.
6. Add 2,5 Volume 100 EtOH, 0,5 Volume 7,5M Ammonium Acetate and 1 µl Glycogen. You don't really need glycogen, however it is pretty handi since you can see the pellet better after centrifugation.
7. Vortex, spin down and incubate 15 to 30 min at -70°C.
8. Prey to the lab-goods and sacrifice something valuable (ramen, pizza voucher etc.)
9. Spin down for 10 min at 13.000 rpm. After that you should see a small white dot (your DNA).
10. Remove supernatant and wash with 50 µl 70% EtOH. Spin down for Spin down for 10 min at 13.000 rpm.
11. Remove supernatant and let the pellet dry.
12. Resuspend the pellet in 40 µl ddH2O. Check the purity and concentration on a gel, using 1 µl of your solution.
Good luck
nice protocol
our easy trap kit is about to finish and new kit not arrived yet,
i can try it sometimes in case of immergency, when the kit will totally finish
how about the yield in this oldfashioned method??? enough for ligation???
I usually made two 50µl PCRs for my PCR product and purified it in one big agarose pocket. The yield was always enough for a standard 10 (or 20) µl ligation.
thats great
work under a hood or a good ventilaed place
1. Cut the band with your product and put it into a tube (you can freeze it at -20°C to make a break or go home to have a life)
2. Add about the same volume of phenol. Use a 1000µl pipette to crush the agarose block as good as possible. The smaller the pieces the better.
3. Vortex like a maniac (3 to 5 minutes)
4. Centrifuge for 10 minutes at 13.000 rpm
5. You should see two phases. The upper one is the aquatious phase and should contain the DNA. Use a pipette to carefully transfer it into a new tube.
6. Add 2,5 Volume 100 EtOH, 0,5 Volume 7,5M Ammonium Acetate and 1 µl Glycogen. You don't really need glycogen, however it is pretty handi since you can see the pellet better after centrifugation.
7. Vortex, spin down and incubate 15 to 30 min at -70°C.
8. Prey to the lab-goods and sacrifice something valuable (ramen, pizza voucher etc.)
9. Spin down for 10 min at 13.000 rpm. After that you should see a small white dot (your DNA).
10. Remove supernatant and wash with 50 µl 70% EtOH. Spin down for Spin down for 10 min at 13.000 rpm.
11. Remove supernatant and let the pellet dry.
12. Resuspend the pellet in 40 µl ddH2O. Check the purity and concentration on a gel, using 1 µl of your solution.
Good luck
Can you use phenol chloroform isoamyl alcohol in place of phenol?
Has anyone had experience with electroelution? I found a paper where they simply cut a small hole in the gel in front of the migrating band and waited for the band to fall in the hole. Then they just pipetted it out and used it directly (no need to clean it up, it's just in TAE). I think it's a specific implementation of the general 'electroelution' method. Other methods use some sort of paper inserted in the gel? I've never tried any of this though.