Hi all,
I am working on a project where we need very high molecular weight DNA for sequencing. We extracted DNA using a Phenol/Chloroform/Isoamyl Alcohol method and then precipitated the DNA using 1/10 volume of sodium acetate and 2 volumes of 100% ethanol. We let the DNA, sodium acetate, and ethanol mixture sit overnight at 4 degrees celsius. We then spun the tube at max speed for 20 minutes, aspirated, and washed with 70% ethanol. After the addition of 70% ethanol, we aspirate the ethanol and either let the sample air dry overnight or used the speed-vac. After using speed vac or letting it air dry, we added 300uL of TE buffer. We let the samples sit at 20 degrees celsius overnight and flicked to mix them a couple times.
In summation- we are unable to dissolve a HMW DNA pellet in TE buffer. We tried heating the sample in a 35 degree water bath for 1 hour, but that still left a very viscous, undissolved pellet. We need high molecular weight DNA and it needs to be in solution. Does anyone have any suggestions?
Thank you.
Dissolve DNA in an alkaline solution may help. The following is the DNA solubilization step from DNAZol protocol.. Dissolve DNA ( without drying } in 8 mM NaOH by slowly passing the pellet through a pippette. Alternatively, dissolve DNA in water. However, the alkaline solubilization of DNA occurs faster and assures full solubilization of DNA precipitate. Add an adequate amount of 8mMol NaOH or water to approach a DNA concentration of 0.2 to 0.3 Aug/Aul. Typically, add 0.2 to 0.3ml of 8mMol NaOH or water to the DNA isolated from 10^7 cells or 10 to 20mg animal tissue. Alternatively, add DNA to 20 to 500 Aug of 10mMol TrisHCl, 0.1mMol EDTA, pH 7.5. Complete dissolution of DNA may require several hours of agitation 37oC or 55oC or may need overnight incubation.
For details consult https://www.protocol-online.org/biology-forums/posts/15163.html
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