How to extract Biotinylated DNA from low melting agarose without use of phenol e - (Jul/01/2015 )
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 doubt biotin will affect precipitation. Not sure about phenol, I haven't used it in years. I would, after ligation, heat inactivate the enzyme and put it thru a G50 spin column. The little fragments should remain in the column.