E. coli DH5 alpha culture problem

shifali chatrath via methods%40net.bio.net (by shifalich from rediffmail.com)
Sat Feb 21 12:50:41 EST 2009



On Sat, 21 Feb 2009 01:18:03 +0530  wrote
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>Dear Members,
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>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 growth rate seemed low. I did not get much pUC19 DNA out of 500 mL culture.
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>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)

Can you please tell the amount of transformation reaction plated? Also, is that you plated everything? 

I have successfully got nearly 1mm  colonies with DH5 alpha!

>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.
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>The growth conditions I used: 
>1)500 mL LB Miller broth in 2-L flask

Can you please tell the composition of LB miller ?( Make sure you read the one written on the bottle from where you took the powder to make medium)

>2) Shaking at 37 C at 300 rpm
>3) 100 ug/mL ampicilin in both LB agar plate and LB broth
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>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 mL culture
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>I know there are a lot of experts on this topic. Please help!
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>Thank you,
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>Sissi
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Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449


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