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Improve expression level in E. coli


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#1 Wang HQ

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Posted 29 March 2002 - 05:59 AM

The expression I am using is pET-30a(+). High IPTG concentration(4mM, 2mM) and 30C did not work well. Although GFP flurescence could be observed SDS-PAGE detected nothing. By the way, Is there codon bias in cotton?

(Edited by Wang HQ at 12:43 am on April 4, 2001)


#2 sean

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Posted 30 March 2002 - 10:48 AM

it really depends on you induction system.

You can improve IPTG induction by increasing the amount of IPTG.

you can also decrease the induction temperature. ( 30' or 25' or even 18')  this helps some people.

sean


#3 milanoj

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Posted 04 April 2002 - 04:15 AM

I'll assume that this fusion is working off a T7 promoter system. This will require 9-12 induction samples. First vary the amount of IPTG 0.1mM, 0.5mM and 1mM and incubate at 37C, 30C and RT. So 3 IPTG concentrations and three temp conditions that's 9 inductions. Keep your antibiotic conc. as high as is practical. lf this is ampR use carbenecillin instead of ampicillin - it's more stable during bacterial growth.




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