Hi All,
I have been trying to extract high quality dsDNA for NGS using phenol-chloroform methods, with and without CTAB and different lysis buffers.
But I always get a huge discrepancy between nanodrop and qubit (dsDNA kits) readings. I recently measured my extracts with the ssDNA kit and found the majority of my DNA (>90%) is degraded to ssDNA.
The DNA is from Cronobacter species with ~52-56% GC, grown overnight (~18hrs). I have used the JGI CTAB protocol and standard phenol-chloroform extractions with SDS lysis buffer. During separations I do not vortex, just invert the tubes several times with ~2sec of a vigorous handshake. The pH of the phenol is stated as >7 but I did measure it with a pH strip which indicates its 7 and could be possibly be slightly less. The phenol is not buffered with tris.
Thanks for the help
I have been trying to extract high quality dsDNA for NGS using phenol-chloroform methods, with and without CTAB and different lysis buffers.
But I always get a huge discrepancy between nanodrop and qubit (dsDNA kits) readings. I recently measured my extracts with the ssDNA kit and found the majority of my DNA (>90%) is degraded to ssDNA.
The DNA is from Cronobacter species with ~52-56% GC, grown overnight (~18hrs). I have used the JGI CTAB protocol and standard phenol-chloroform extractions with SDS lysis buffer. During separations I do not vortex, just invert the tubes several times with ~2sec of a vigorous handshake. The pH of the phenol is stated as >7 but I did measure it with a pH strip which indicates its 7 and could be possibly be slightly less. The phenol is not buffered with tris.
Thanks for the help
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