Lactococcal plasmid isolation

Morris Waskar mwaskar at yahoo.com
Tue Mar 26 04:15:09 EST 2002


Hi everybody,

We have been trying to isolate plasmid from a bacteriocin producing
Lactococcus lactis culture. Repeated isolation using the method of DJ
O'Sullivan and TR Klaenhammer (Appl. Envir. Microbiol. 1993) have
yielded preps with plasmid apparently not able to move into agarose.
The maximum it moves in a 0.7% agarose gel in 0.5X TBE is less than 1
mm after 4 hrs at 100V, even in the presence of SDS. After treatment
of the crude plasmid prep with papain, the plasmid enters agarose gel
and runs quite well ( more than 2cm in an hr at 50V), but a
phenol-chloroform or a chloroform step to remove papain seems to
remove the plasmid and the yield decreases as much as 90% at every
step. We have even tried to introduce papain and Proteinase K in the
earlier steps but we still land up with a high molecular weight
complex. Can anyone suggest any additions/ modifications or alternate
protocols? Thanks for the help.

Morris




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