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Design PCR primers for cloning 3 copies of same insert

PCR Primer design cloning

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#1 HawkeyeGrad

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Posted 16 September 2013 - 07:52 AM

Hi All,

I want to clone 3 copies of a DNA fragment into an expression vector and was wondering if anyone has any insight on primer design. I've researched a few cloning protocols but the main question is this: Since the DNA sequence fragment is the same, the primers are technically going to be the same and I would have a problem with primer dimers. How can I deal with the dimer issue and get specific primers made? Any help with this is appreciated!

 

Thanks!



#2 phage434

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Posted 16 September 2013 - 07:58 AM

Primers and repeated sequence go together poorly. You would have better success using restriction digests. A restriction digest on both ends of your fragment, which is removed by ligation can pair fragments. A subsequent reaction will add a third. With offset cutters you can do this with an arbitrary sequence.



#3 HawkeyeGrad

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Posted 16 September 2013 - 11:03 AM

I forgot to mention, I already have one sequence in the destination vector and want to add 2 more to the vector. Is there an easier way in which I can clone the fragments in all at once in stead of performing multiple digestions and ligations? From my understanding, I could add degenerative sequences to my fragments along with restriction sites then cut and ligate the fragment together. Should the restriction enzymes be different for each end of the fragment and/or each fragment?







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