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cloning


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4 replies to this topic

#1 sdar

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Posted 09 February 2010 - 06:27 AM

i want to clone a gene into expression vector for expression in bacteria my gene has got 10 stop codons in the middle of the gene that doesnot allow my protein to be fully expressed. what to do?

#2 pnjn

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Posted 09 February 2010 - 12:06 PM

I am sorry this is not the reply but I will be thankful to u if you could pls tell me how to send a post in general.

#3 bob1

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Posted 09 February 2010 - 04:18 PM

10 stop codons in the gene? Are they in-frame?

#4 HomeBrew

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Posted 09 February 2010 - 05:44 PM

If they are in frame, I don't know what you have, but it isn't a gene. If they're not in frame, they are of no consequence.

#5 fishdoc

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Posted 09 February 2010 - 06:57 PM

A colleague of mine had a similar problem with a not-too-commonly studied bacterium. There were 3 or 4 stop codons (for E. coli) in the ORF that actually encode an amino acid in the particular organism rather than causing a stop. They didn't realize that cloning it into E. coli would result in a truncation at first. Once they did, they ended up having the gene made synthetically with each of the stop codons modified at the wobble position so that the correct amino acid is added and the stop is removed. Thus far, the synthetic hasn't expressed, but I have a feeling that's more of user error than a sequence problem.

Of course, having an entire gene made synthetically can be expensive. But your other option is to make a point mutation at each stop codon to change the wobble position so that you can clone into E. coli and get the correct amino acid.




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