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cloning of race products


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#1 deespike

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Posted 20 March 2011 - 06:45 AM

Hi all

I am trying to perform 3' race and am using invitrogen kit. I got amplification using my gsp and kit supplied UAP . I am not sure now whether I can clone it into ta vector. The presence of uracil is going to affect or not.
please help

#2 Trof

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Posted 20 March 2011 - 10:08 AM

What polymerase did you use to amplify your product? It should be Taq-based, or other polymerase that adds "A" to the end of the amplicon (check the manual), otherwise you can't TA clone it. Also use as fresh product as possible, terminal "A"s tent to degrade quickly.
I don't understand what uracil you're talking about, uracil is in the RNA, and you had to RT the transcript and amplify it, unless it's a kit with dUTP (to prevent cross-contamination), there should be no uracil. And if that is the case I don't think it would interfere with cloning, but E.coli cells (says Wikipedia)contain UNG that cuts off product with dUTP in it.

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