Episome Transfer

Rafa Maldonado rafael at genetics.med.utah.edu
Tue Jan 11 21:20:18 EST 1994


Hi Seth
I suppose your episome is a F plasmid, because you mention that your
recipient strain is F-; if not, correct me.
First of all, F plasmids are too big to be isolated by a standard
plasmidprep, so you can't do the alkaline miniprep and electroporate.
Besides, the electroporation (and transformation in general) frequency
decreases with the size of the plasmids, and I don't think that a 60Kb
plasmid can achieve a modest frecuency to be transfered in that way.
The easiest way is a mating between your F+ and your F-, because F is
an auto-transmisible plasmid. This can be done mixing a culture of each
strain (explained later).
But you need a system to select against the donor and the recipient
that didn't receive the episome. You told me that your episome carries
a Kan resistance gene, so after the mating you can select against the
recepient that didn't receive the plasmid with kanamycin. To select
against the donor, you need one of this things:
1.-A antibiotic resistance gene resistance in the recipient, or
2.-A mutation in the donor so it cannot grow in some media; for
example, if the donor is auxotroph for something or it cannot use some
sugar, you can use minimal media without the requirement or with the
sugar as carbon source.
If your recipient strain doesn't have any of this things, you can
select a antibiotic-resistant (i.e. nalidixic acid) derivative of it.
You can try to get the mutation spontaneously (spread 0.2 ml of
overnight culture in plates of LB+nalidixic acid 30ug/ml).
The protocol is from: JH Miller (1972) Experiments in molecular
genetics; Cold Spring Harbor Press:
1.-Subculture overnights by diluting 1:40 of both recipient and donor
and grow at 37 until the donor is 2-3x10(8) (usually three hours).
2.-Wash the donor cultute if you used antibiotic: centrifugue the
culture and resuspend the cells in LB without antibiotic.
3.-In a well dried LB plate, put a drop (aprox 30 ul) of the donor, a
drop of the recipient and a drop of previously mixed donor and
recipient in ratio 2:1.
4.-Let dry the drops and incubate 37 overnight
5.-Take some cells of the mating drop ans streak in the selective
medium (antibiotic o minimal medium, with kanamycin to select the F).
As control, streak in other plate the donor and the recipient drops.
Also, you cam replicate the mating plate with velvet onto the selection
plate.
I don't know the posibilities of selecting against the donor you have.
If you need more help, message me.
WARNING! If your plasmid is not F, this system may not work! Some
others plasmid are self-transmisibles too, but other not. Please, ask.

Rafael Maldonado
HHMI-University of Utah
Salt Lake City, Utah
rafael at genetics.med.utah.edu



More information about the Methods mailing list