Hi,
I am trying to remove unligated, biotinylated linkers/adapters from my DNA by running a low melting point agarose gel (0.8%). After cutting the DNA out, and leaving the unligated small sized linkers on gel, I am trying to do a gel extraction. I have already tried Qiagen gel extraction, Silica beads. Some of the experimental facts are:
1) The linkers are ~25 bp, so easy to separate. But the target DNA is a smear ranging from ~100 bp to 10kb. So I am running the gel for short duration, enough to see the bright band of unused linkers separated from rest of the DNA
2). The target DNA will also be biotinylated now because of the ligation.
Due to low yield (only 2ug recovered from a 20 ug startup DNA), I am thinking of dissolving the agarose piece in Tris and do a Phenol extraction followed by EtOH precipitation. My question is how will phenol affect the biotin, will it still be able to bind to streptavidin? Also, will it be easy to precipitate biotinylated DNA or will the biotin give buoyancy to the DNA so that it is hard to precipitate now?
i do not know the effect of phenol on biotinylated dna but in answer to your second point I would expect your dna to spin out very well but if you are worried about a small residue not spinning down you could add glycogen or some rna to bulk up the centrifuged precipitated dna
Thanks Paul. I guessed using Glycogen or other carriers should precipitate the biotinylated DNA. However I am not still clear about whether to use phenol extraction or not.
if nobody comes up with a definative answer you can always dissolve the agarose away using agarase to break it into monomers and precipitate your dna out of that
Hi Paul, you mean dissolving the low melting point agarose and directly setting up the ethanol precipitation without phenol extraction?
Now I am wondering what exactly the phenol extraction does? is it to completely remove the agarose?
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