I would like to know whether it would be possible to prepare(extract) DNA from high molecular weight DNA which was embedded in agarose plugs (please see the method in Osoegawa K, Woon PY, Zhao B, Frengen E, Tateno M, Catanese JJ, de Jong PJ: An improved approach for construction of bacterial artificial chromosome libraries. Genomics 1998, 52:1-8.). I do have several plugs having been prepared in 2010 and it would be great if we could use them for DNA to be used for NGS (by HiSeq Illumina)sequencing project could you please help?
yes you can. you may either dissolve the agarose with agarase and precipitate the dna or melt the plug in a few volumes of water and extract as usual
Thank you for the answer Paul. The plugs are in a solution of 0.5M EDTA and we thought of melting them as in your second suggestion followed by phenol extraction and precipitation. Is there any particular reason you would dilute it in water instead of EDTA?
Razvan
Not really Razvan . Water is very flexible as to what you can do next and dilutes salts and any inhibitors. 0.5M edta seems very strong and would chelate magnesium so probably agarase would not work as most enzymes need a divalent ion to work properly and I would probably dissolve in something to lower the salt concentration but TE is a good buffer and enzyme inhibitor so is probably the diluent of choice prior to extraction.
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