Methods Digest, Vol 53, Issue 1

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Fri Oct 2 07:15:50 EST 2009


I am surprized by what you are observing. Keep one thing in mind. pUC19
transformed pure clones do not differentiate between LB or LB amp. The
plasmid does not provide anything for higher growth of the bacterial cells
as it will be same in LB or LB-amp. Plasmid allows only selective growth of
transformed cells in LB-amp and not the higher growth. You should get same
growth rate of pure culture in Lb as well as Lb-amp.

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> Kyle
>
> Thank you for the advice. I am still trying to figure out why my culture =
is
> not growing well.
> Now
> I just test the starter culture since it did not grow to the expected
> cell densities in the first place. The OD600=3D0.75 after 20 hr
> incubation at 37C at 300 rpm. The culture was half transparent after 20
> hrs growth.
>
> Here was the procedure:
> The colony was picked from fresh agar plate.
> The single colony (around 1.2 mm in diameter) was inoculated to 2.5 mL LB
> broth (with or without Amp)
> After 20 hrs growth, both tubes gave me the same cell density, OD600=3D0.=
75
> (with or without 100 ug/mL Amp).
> I checked the pH of LB broth and adjust it to pH7.5 before autoclave.
>
> I
> really do not know what the trouble is. Transformed DH5 +pUC19 should
> grow very fast. I just placed an order for liquid LB broth from
> different vendor (not dehydrated LB), in case the problem lies in the
> water I used to make LB broth or something. Could it be contamination
> issue? But I don't see  how.
>
> The super broth formula you used seems very good. I will try it if the ne=
w
> LB broth I just ordered will not work. Thanks, Sissi
>
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--=20
Dr V K Gupta
Sr Microbiologist (Molecular Biology)
Insect Molecular Biology Lab
Department of Entomology
Punjab Agricultural University
Ludhiana (Pb)-141004- India
M: 09815963210


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