I'm new to the field of PFGE and have a few questions before I begin the experimental procedures. When I separate out my DNA size of interest, how can I purify it from the gel without using a purification kit? My fragment size is >50 kb and most of the commercial gel extraction kits recommend using their kit if the fragment to be separated is up to 10 kb. Also, when I purify DNA from the gel, is what I obtain just DNA, i.e. histones and other proteins are washed away? What if I cross-link protein-DNA complex and run that on the gel? Ultimately, I'd like to do a histone ChIP experiment on just my fragment of interest, but I don't know enough details of PFGE to go through with this experiment. Any suggestions would be appreciated. Thank you!
You can purify the DNA by agarase treatment (see Santos et al. (2010) Environ Microbiol 12:2965–2976). If the EPS treatment is complete, there should not be proteins bound to the DNA. Good luck!
- Firstly : the genomic DNA is from bacteria? if yes, so you have to know that DNA of bacteria do not contain Histone proteine ( the case in Eucaryote DNA), so be sure that there is not proteine that migrate with your DNA in agarose gel.
Secondly: You can cut the fragment of gelose containing DNA that you want, pout this fragment in an eppendorf tube containg coton. then cut a smoll whole in the bottom of this tube. Then take another eppendorf tube, in which you pout the first tube; then centrifuge a 1000 tr/min during 5 minute. By this whay the liquide of agarose containg DNA is recuperated in the tube (2) and the agarose remain in the first tube. You can then precipitae DNA by two volume of absolute DNA, I explain : for exemple if you recuperate 100 µl of agarose yopu so add 200 µL OF ETANOLE,
You can also cut the band of interest and perform an electrophoresis on dialisis tubes; you can find the protocol on the Maniatis. In any case, whatever the protocol you use, you should do a purification step (phenol-chloroform extraction, column purification, etc.), which will remove all proteins bound to the DNA. I you cross-lind DNA with a protein, migration will be reduced, as it happens with the shift mobility assays.
if your interest is DNA with length more then 50kb you should prepare plags from your samples. All other method degrad DNA.
electrophoresis in glass tubes is besr chois for isolation long DNA fragments from agarose
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