E. coli DH5 alpha culture problem

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Tue Feb 24 08:54:37 EST 2009


On Feb 20, 12:42=A0pm, Sissi zhang <sissi_2... from hotmail.com> wrote:
> Dear Members,
>
> I need quite a bit of pUC19 vector, so I transformed it into DH5 alpha an=
d grew the culture. I run into a problem with DH5 alpha culture. The growth=
 rate seemed low. I did not get much pUC19 DNA out of 500 mL culture.
>
> Here are the things I observed:
> 1) Transformation efficiency was good, reached 10^7, however, the colonie=
s were very small after overnight growth on selective LB Miller agar plate =
(less than 0.5 mm in diameter)
> 2) Starter culture gave OD600=3D0.5 after 8 hours growth (a single colony=
 in 2 mL LB, Miller broth with 100 ug/mL ampicillin)
> 3) 2nd culture: 1 mL of starter into 500 mL LB Miller broth with ampicill=
in
> =A0 =A0 OD600=3D0.9 after 12 hours growth
> =A0 =A0 OD600=3D1.15 after 16 hours growth
> =A0 =A0 OD600=3D1.34 after 18 hours growth
> 4) Cell pellet wet weight after 18 hours growth was 1 g from 500 mL cultu=
re.
>
> The growth conditions I used:
> 1)500 mL LB Miller broth in 2-L flask
> 2) Shaking at 37 C at 300 rpm
> 3) 100 ug/mL ampicilin in both LB agar plate and LB broth
>
> Questions:
> 1) Do you think the growth rate was slow?
> 2) If so, what could be the reasons? I heard that DH5 alpha tend to give =
small colonies, even so, why the total yield was so low? I guess I could le=
t the colonies continually grow to much bigger colonies before liquid cultu=
re. If I start with a much bigger colony, would I get OD600=3D4.5 after 16 =
hours growth(this growth rate is shown in Qiagen plasmid DNA purification b=
rochure)?
> 3) pUC19 should be high copy vector but I got only 0.6 mg DNA out of 500 =
mL culture
>
> I know there are a lot of experts on this topic. Please help!
>
> Thank you,
>
> Sissi
>
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Well, you have to seed pre-culture single colony in say 5-10 ml LB
+Abt, and then innoculate the 500 flask.
my2c


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