Methods Digest, Vol 45, Issue 17

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Tue Feb 24 08:06:14 EST 2009


Some points need to be clarified. starter culture (if it is DH5 ?)- you have
been addding ampicillin which inhibited growth.
It is also possible that puc 19 is defective for gene that controls high
copy number. Acquire puc 19 from some other souce or try with some other
plasmid. This will help in standardizing transformation conditions.
Streak DH5 culture on LB agar, isolate well growing colonies and streak on
LB  and LB ampicillin plates. Select clones that are growing on LB and not
on LB ampicillin for transformation. Hope it will solve.


On 2/22/09, methods-request from oat.bio.indiana.edu <
methods-request from oat.bio.indiana.edu> wrote:
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>   1. Re: E. coli DH5 alpha culture problem (shifali  chatrath)
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> ----------------------------------------------------------------------
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> Message: 1
> Date: 21 Feb 2009 17:50:41 -0000
> From: "shifali  chatrath" <shifalich from rediffmail.com>
> Subject: Re: E. coli DH5 alpha culture problem
> To: <sissi_2009 from hotmail.com>
> Cc: methods from magpie.bio.indiana.edu
> Message-ID:
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> 1235159283.S.4403.11701.f4mail-235-204.rediffmail.com.old.1235238641.16613 from webmail.rediffmail.com
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> Content-Type: text/plain; charset="ISO-8859-1"
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>
> On Sat, 21 Feb 2009 01:18:03 +0530  wrote
> >
> >
> >
> >
> >
> >
> >
> >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 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 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.
> >
> >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
> >
> >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
> >
> >I know there are a lot of experts on this topic. Please help!
> >
> >Thank you,
> >
> >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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> ------------------------------
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> End of Methods Digest, Vol 45, Issue 17
> ***************************************
>



-- 
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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