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E. coli DH5 alpha culture problem

E. coli DH5 alpha culture problem - Protocols and Methods Forum

E. coli DH5 alpha culture problem - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 02-20-2009, 05:42 PM
Sissi zhang
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Default E. coli DH5 alpha culture problem










Dear Members,

I need quite a bit of pUC19 vector, so I transformed it into DH5 alpha and grew the culture. I run into a problem with DH5 alpha culture. The growthrate 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 colonies were very small after overnight growth on selective LB Miller agar plate (less than 0.5 mm in diameter)
2) Starter culture gave OD600=0.5 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 ampicillin
OD600=0.9 after 12 hours growth
OD600=1.15 after 16 hours growth
OD600=1.34 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:
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 let the colonies continually grow to much bigger colonies before liquid culture. If I start with a much bigger colony, would I get OD600=4.5 after 16 hours growth(this growth rate is shown in Qiagen plasmid DNA purification brochure)?
3) pUC19 should be high copy vector but I got only 0.6 mg DNA out of 500 mLculture

I know there are a lot of experts on this topic. Please help!

Thank you,

Sissi



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  #2  
Old 02-21-2009, 02:50 AM
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Old 02-24-2009, 01:54 PM
peter
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Default E. coli DH5 alpha culture problem

On Feb 20, 12:42*pm, Sissi zhang <[Only registered users see links. ]> wrote:

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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Old 02-28-2009, 08:09 AM
Kyle Legate
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Default E. coli DH5 alpha culture problem

peter wrote:


I agree, the seeding culture was probably too small and not ready to add
to the 500 mL culture, resulting in an unacceptably long lag phase. It
should be 5-10 mL as Peter said and you should not be able to see
through it when you add to the larger flask. Some people spin down the
starter culture and resuspend in fresh LB-Amp to remove the secreted
beta-lactamase but personally I don't do this for plasmid amplification.
The long lag phase you experienced due to a dilute seed culture
containing beta-lac may have depleted the antibiotic, resulting in some
of the bacteria losing the plasmid, resulting in low yield.

For a high yield of plasmid you can grow DH5a in Superbroth: 32g
tryptone, 20g yeast extract, 5g NaCl, 1.25mL 4M NaOH per litre. I
routinely get >1 mg of DNA per 100 mL of culture with this recipe.
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