I have a gene I've constructed in 3 pieces into a petblue2 vector. When I try and PCR out the gene I get a band ~4kb (size of vector and insert). When I aligned the sequences of my primers and the vector only 30% max overlaps. What is the minimal % overlap that would lead to the template being aplifed? I'm trying to figure out how to fix this.
Currently, my plan is to order the primers that will PCR out the MCS with my insert and use this product to amplify my gene. I need to get it out of this product. I have a 27 BP overlap with my product on each primer to get a Tm of 65 and still get the 4kb+ nonspecific band and no band for my product.
Minimum Primer Overlap for Product?
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