can agarose be removed by phenol? - (Jan/31/2007 )
recently, I saw a protocol discribe a method which extract nucleic acid directly from the agarose gel by phenol: digested-elecrophoresis-cut the band you want-put it in a EP tube-crush by a tip-add 500 microliter H2O,shake- add 500 microliter tris saturated phenol,shake,centrifuge-take the water phase,add chloroform,shake -centrifuge, take the water phase, add NaAc, and EtOH to precipitate the nucleic acid.
The protocle sounds so reasonable,but there is one point i dont understand:can the agarose be remove by the phenol? whats the mechanism behinde it?
thank you for any tips and sugestions.
Agarose is a polysaccharid. I think the idea behind the protocol is, that phenol-chloroform can be used to separate nucleic acids from proteins, lipids and polysaccharids. But I don't know enough chemistry to explain the mechanism behind it...
However, I used a similar protocol once, it worked, but I prefer a column based extraction to avoid too much phenol-chlorphorm work...
I am not too sure. But I thought you can actually replace the column with a normal filter paper. Once you dissolve the agarose gel, run it through filter paper. I thought the filter paper will hold the agarose and the solution with dissolved nucleic acid will travel down.
i think one thing may be add to your rotocol. You may heat to 50° ypur water + agarose to enhance dissolutionn of agarose.
The water:agarose ratio should be very important as agarose tend to gelify at lower temp when dissolved.
After heating to 50° cool 2' at 37 and then add phenol and vortex well
I remember you need to melt it, add some 5 M NaCl to it then extract with phenol. But I like this way: stuff some glasswool to a 1 ml tip cut to fit in a microcentrifuge, add gel blocks inside the tip, freeze in -80 C freezer, spin 5 min top speed, then quickly remove the liquid. Simple and fast.
Recently I found this protocol. Sounds good... anyone has experience with it or something like?
*Phenol Purification of DNA From Low Melting Point Agarose*
Prepare phenol mix: 25ml pure phenol, 15ml H2O, 400ml 1M Tris-HCl, pH to 8.0 with 2 drops 5M NaCl
1. Place phenol mix at 37oC for at least 1 hour
2. Melt excised agarose fragment at 65oC. for 10 minutes or until agarose is molten, then place molten agarose at 37oC for 2 minutes
3. Add 0.5X volume of phenol mix and shake vigorously for 45 seconds
4. Centrifuge for 5 minutes at room temperature and transfer aqueous layer to clean tube
5. Repeat phenol extraction and centrifugation step
6. Transfer aqueous layer to a clean tube and add 1/50 volume of 5M NaCl, shake for 45 seconds, and centrifuge for 5 minutes at room temperature
7. Transfer aqueous material to a clean tube, add 2X volume of EtOH and precipitate (either 15 minutes at -70oC. or at least 1 hour at -20oC)
8. Centrifuge for 15 minutes and aspirate off aqueous material
9. Wash pellet in 1X volume of 70% EtOH and repeat centrifugation
10. Aspirate off aqueous material and air dry pellet for 5 minutes (or dry in speedvac for ~2 minutes)
11. Resuspend pellet in 20µl TE buffer