The protocol just deals with the bare bones of the digest, T4 DNA polymerase treatment, annealing and transformation. For information in designing the cloning site, see my original article, or the paper.
I think we all have been through those my-PCR-product-didn’t-get-amplified days. Sometimes, playing around a bit more with the PCR-conditions brings luck, or sometimes it doesn’t work at all. These days we have access to many different types of DNA polymerases, ultrapure and buffered nucleoside triphosphates, and other necessary starting materials in convenient concentrations; but still […]
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