Michael W. Thompson
mthom0 at pop.uky.edu
Fri Dec 20 12:09:13 EST 1996
Chi-kuang Wen wrote:
> Rajesh Kumar (rk6n at VIRGINIA.EDU) wrote:
> : Hi Netters,
> : I am screening a genomic library made up in lambda ZapII vector. I want to
> : know the growth conditions so as to get 20 000 plaques per 150mm plate.
> : Right now I am following Maniatis (NZY plates, plaques are grown for 12h
> : at 37C), but I do not get more than 2 000 plaques (almost touching each
> : other) per plate. The size of my plaques is almost equal to a well grown
> : bacterial colony.
> : Any suggestions in this regard will be appreciated.
> Dear Rajesh:
> First, you may want to check the titer of your library.
> Also, the bacterial strains might be important for some genomic DNA which
> contains hyper-methylated DNA. If the strains were not right, then you
> won't get a good titer.
> Third, plating the phages is important, if your top agarose partially solidifies, your titer will be highly reduced. To check this, just see if the top agar
> is smooth after it's solidified.
A few more suggestions: Make sure you include Mg in the reaction
mixture when you adhere your phage to your host cells. If I use 300 uL
of host cells, I add the correct phage dilution in an equal volume of SM
2nd> Don't use top agar. Top agarose is much better.
3rd> The temperature you keep your top agar/agarose at before plating
is critical. If it's too high, your bacterial cells will die and will
grow very slowly. I use 47C. Up to 49C would probably be OK.
4th> You might check and make sure that the host strain you're using is
the recommended strain from that library (check with the manufacturer).
In addition, if you grow up cells beforehand, it's best to use them
within 24 hours, and to grow up new plates of host cells about every
week or so.
Hope this helps too :)
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