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Do genes always need to be cloned in the "right direction"?


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#1 rosephish

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Posted 26 September 2013 - 12:37 PM

Greetings all,

 

I am doing a cloning project where I'm inserting a 2kb gene fragment into 2 different plasmids; for one of the plasmids, the restriction sites are located such that the 5'->3' direction of the gene is in the same direction as the transcription direction arrows on the plasmid. But for the other plasmid, the gene would be going "opposite" the direction of the arrows. I was able to easily clone the gene into the "same direction" plasmid, but have yet to get any luck with the "opposite direction" plasmid, I was wondering if the directionality is why it's not going in?

 

PS--My boss is the one who designs the primers, I haven't learned how to do all that yet; I just work with what I'm given.

 

Thanks for any help, I've been registered here for a while, but this is my first time posting smile.png


Edited by rosephish, 26 September 2013 - 02:46 PM.


#2 theo22

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Posted 26 September 2013 - 01:36 PM

Do you want to have your gene expressed from the plasmid? Then the gene needs to be cloned behind the promotor. So yes, then the orientation is important. When you talk about "replication arrow" do you mean transcription direction?



#3 rosephish

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Posted 26 September 2013 - 02:45 PM

Yes, the transcription direction, thanks; and I don't think we need expression, but I'm not 100% certain on that; but it sounds like maybe if we don't need expression, then orientation shouldn't matter in regards to getting it cloned into the vector; in which case, darn, not sure why my cloning isn't working. Most of the time I can clone into the various plasmids we use quite easily, but this construct just doesn't want to cooperate :)

 

Thanks for the help!



#4 phage434

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Posted 26 September 2013 - 04:20 PM

If you are cloning a protein sequence toxic to the cell, then cloning in the direction of transcription can result in no growth. Cloning in the other orientation, which will not be expressed, is then much easier.






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