E. coli DH5 alpha culture problem

Sissi zhang via methods%40net.bio.net (by sissi_2009 from hotmail.com)
Fri Feb 20 12:42:02 EST 2009








Dear Members=2C

I need quite a bit of pUC19 vector=2C 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=2C reached 10^7=2C however=2C the col=
onies were very small after overnight growth on selective LB Miller agar pl=
ate (less than 0.5 mm in diameter)
2) Starter culture gave OD600=3D0.5 after 8 hours growth (a single colony i=
n 2 mL LB=2C Miller broth with 100 ug/mL ampicillin)
3) 2nd culture: 1 mL of starter into 500 mL LB Miller broth with ampicillin
    OD600=3D0.9 after 12 hours growth
    OD600=3D1.15 after 16 hours growth
    OD600=3D1.34 after 18 hours growth
4) Cell pellet wet weight after 18 hours growth was 1 g from 500 mL culture=
.

The growth conditions I used:=20
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=2C what could be the reasons? I heard that DH5 alpha tend to give =
small colonies=2C even so=2C why the total yield was so low? I guess I coul=
d let the colonies continually grow to much bigger colonies before liquid c=
ulture. If I start with a much bigger colony=2C would I get OD600=3D4.5 aft=
er 16 hours growth(this growth rate is shown in Qiagen plasmid DNA purifica=
tion brochure)?
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=2C

Sissi



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