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Genomic Library Creation -- Vector recombination problems

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#1 tatjr13



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Posted 03 November 2015 - 12:45 PM

So I've been trying to make a genomic library from a type of bacteria and the method I've been using is the following: 


-Digest the genome with a 4 cutter enzyme (Sau3A1) -- cuts at GATC

-PCR amplify my vector and digest to create the appropriate sticky ends BamHI (G^GATC_C) and BglII (A^GATC_T)

-Treat the vector with shrimp alkaline phosphatase to dephosphorylate and prevent recombination

-Ligate the insert/vector together overnight at 16C


When doing this I found that all of my clones (confirmed by sequencing) were recombinations of just the vector. 


Is there something I'm missing? Does SAP not dephosphorylate PCR products? I'm choosing to PCR amplify the vector instead of digesting simply because it provides you with a higher concentration of DNA to work with during cloning (my vectors are low copy number).


Any help is much appreciated! 





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