This is really newbie stuff I am asking.
Besides a control to check polymerase function, I am setting up four OE-PCR reactions with different ratios of the two molecules: [Molecule 1] the insert-flanking-vector-sequences linear molecule, and the [Molecule 2] very common vector molecule (pUC19). The ratios are 50:1, 100:1, 250:1, and 500:1.
Path 1: After the amplification and the digestion with DpnI, is there enough product that can I take an aliquot of each reaction, digest with two restriction enzymes (in my case AlwNI and SspI) to look for expected bands on agarose gels? After that, I would then transform cells with the remainder.
Path 2: Or should I take all the post-DpnI-digest reactions, do the transformations followed by plasmid minipreps and characterize the miniprep product?
By the way, I am choosing 18 cycles on the OE-PCR, suggested as optimal in the article, although the recommendation is 5-30 cycles to be tested. I suppose if the 18 cycles fails, I could pick a 250:1 ratio at 5, 12, 20, 30 cycles. Is that a good strategy for those with experience?