David,
-- If you don't have access to a uv spec then you can cut your plasmid as Dakota suggested. Use enzymes that give you a fragment size similar to your expected pcr product - 6400bp. For example, XbaI/EcoRV will liberate a 6,593bp fragment based on Tom's sequence - AF170104 (don't use this cut as pcr template!). Run it with a known amount of 1kb ladder. You can estimate how much each run of the ladder is based on how much you add. See the manufacturer's instructions. This will give you an estimate of how much DNA you have.
Yes, evaporate the ethanol. It will cause problems for you!
XbaI looks unique in the plasmid so I would cut your template with that first before doing the PCR. It doesn't fall within your target.
Dakota, the primers do look right compared to Tom's sequence.
Cheers,
Scott
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