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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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why
Posted by: popogirl (IP Hidden, New member, 9)
Date: March 4, 2006 06:39PM
Hi, everyone:
I am making a recombinant protein(55KDa). I clone it into PET28+plasmid(Karnamycin resistant). I have it sequenced and the result is perfect. So I try to express it in BL21. In the first time, after IPTG induction for 4h, I got very big induced bands on gel but when I repeat it for second time and third time, I lost the induction completely. I really don't know what is going on. Please help me figur out the problem. Thank you.
Re: why
Posted by: elixir (IP Hidden, New member, 3)
Date: March 7, 2006 09:48PM
Hi,
Check your glycerol stocks, as in do a mini induction of the glycerol stocks and check whether they are working fine... Most of the times you will get induction when you pick a colony directly out of a plate , but not from the glycerol stock!..This means that you need to keep atleast 5 copies of glycerol stock and keep checking its viability. You can replate your glycerol stock and make fresh ones if necessary.. Hope this helps.
Re: why
Posted by: supernov (IP Hidden, New member, 3)
Date: March 24, 2006 05:34AM
True, I had these problems with GateWay-system inductions. What I'm doing now is transforming fresh bl21's every time, growing them o/n on agar-plates and use them to do an overday induction, straight from the plate. If I do an overnight prime-culture, dilute it the next day and try to use it for my induction, it also fails...makes no sense to me, but it's how it goes. :)
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