I would like to prepare mono nucleosomes for my single molecule experiments.
We have PGEM 3Z vector (size is 3kb) from which we are amplifying the 344bp oligo that contains the Widom 601 sequence which is required for preparation of nucleosome. After PCR amplification we see that there is a significant amount of vector in the sample which was observed in gel electrophoresis. How do I obtain pure 344bp oligo?
And also please let me know if the purity of 344bp DNA that I am using for this purpose is crucial for reconstitution.
Yes, the purity of the DNA template is important for reconstitution.
First, you can separate the DNA template from the rest of the vector by PEG precipitation. Here is a very old paper that describes the principle: Lis & Schleif, Nucleic Acids Res, 1975 "Size fractionation of double-stranded DNA by precipitation with polyethylene glycol". Basically, larger DNA fragments should precipitate at lower PEG concentrations. Thus if you do a careful titration with PEG (usually 4-10%), your large DNA vector will precipitate earlier than the small DNA fragment, meaning that you can separate them.
Additionally or instead of the PEG precipitation you can try using the anion exchange column and high-salt elution gradient. There larger DNA fragments should in principle elute later - at higher salt concentrations.
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