I want to do the inverse PCR using 5’ phosphorylated primers to get my interested deletion constructs by deleting (skipping) the internal region.
I have already phosphorylated the primers with T4 PNK, but I don’t know the further steps after performing the PCR. Should I directly add DpnI to the PCR product? How about the self-ligation of the PCR product (blunt-end cDNA) with 5’ phosphorylation? Is the product going to be auto-ligated or that I should treat it with T4 ligase? Should I purify the PCR product?
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