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Gibson/SLIC question


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#1 Rsm

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Posted 05 March 2014 - 05:52 AM

I'm a bit confused regarding Gibson assembly or SLIC. Using an exonuclease should result in regression of both 5' ends, right? Why is in all schemes only one of them degraded ( https://www.neb.com/...bly-cloning-kit )? Isn't there a chance that the whole PCR product will be eventually degraded? Or are they using modified primers?

 

Thanks,

 

rsm


Edited by Rsm, 05 March 2014 - 05:55 AM.

I got soul, but I'm not a soldier

#2 Rsm

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Posted 07 March 2014 - 12:01 AM

Sure, that makes sense. What I mean is that both ends of the vector and the PCR product should be chewed away, such as

 

5'--------------------------------3'        goes to                      5'-----------------3'        and not    5'-------------------------3'      as they show in the picture. 

3'--------------------------------5'                           3'------------------5'                                      3'-------------5'

  

Why is the left arm of the DNA "protected" in the pic? ie why is only one DNA strand degraded?


Edited by Rsm, 07 March 2014 - 12:02 AM.

I got soul, but I'm not a soldier




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