JHartley at lifetech.com
Wed Oct 25 06:09:08 EST 2000
You might want to save 500 ul or so of your cleared lysate before you put it
on the column. Precipitate with 2 vols of ethanol or 1 vol isopropanol,
dissolve in TE, apply to gel next to supercoiled stds. Apply what you
recover from the column in an adjacent lane. Some crude calculations of the
volumes will tell you whether your losses came before or after the column.
Do you need to use a purification column? They may remove some protein,
although there is almost none to begin with, and most, but not all, of the
RNA (really overload a gel and you'll likely see some). But they don't
remove chromosomal or nicked or dimer plasmid as far as I can tell. Perhaps
you should consider direct alcohol precipitation of the cleared lysate.
FWIW, all the standard cloning E. coli strains are endA minus, which means
that phenol extraction of alkaline lysis DNA is unnecessary, at least to
protect the DNA. Try doing a RE digestion with and without a phenol
James L. Hartley, Ph.D.
Life Technologies / Invitrogen
PO Box 6482
9800 Medical Center Drive
Rockville, MD 20849-6482
jhartley at lifetech.com
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