Chloramphenicol plasmid amplification.

Matthew L. Brown, Ph.D gemini3 at mindspring.com
Wed May 7 16:54:14 EST 1997


Stuart,

	I chloramphenicol boost my cultures containing pCRII routinely to get
good yields. I grow first in LB + Amp (50 - 100 ug/mL) until the density
(Absorbance at 600nm) is about 0.6 to 0.7 (about 4 or 5 hrs) then I add
chloramphenicol at a final concentration of 200 ug/mL and continue
incubation overnight(all at 37 C). This seems to work fine for me. My
understanding of the principal is that with low copy number plasmids you
have to use so many cells to get a reasonable mass of plasmid that the
protein in the cells interferes with most plasmid prep methods. By
letting the cells grow to a reasonable level and then adding
chloramphenicol (a protein synthesis inhibitor) at a sublethal level you
allow DNA synthesis (and plasmid replication) to continue at a normal
pace within cells that are dividing more slowly (due to inhibition of
protein synthesis). Therefore, fewer cells in the end with more copies
of the plasmid per cell. As always, feel free to correct me if I'm
wrong.

Good Luck,

Matt Brown, Ph.D.
Dept. of Medicine
Univ. of Alabama at Birmingham



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