If the volumes you listed before are correct you have a final concentration of 1uM primers which is fine.
I've not checked your primer sequences so I don't know if they actually anneal to anything in your target. Did you design them yourself? Get them from a paper?
Run a few microliters of your miniprep and you should see your plasmid, make sure it's cleaned up well.
On Tue, Mar 1, 2016 at 11:59 PM, David Ishee <midgardkennels@gmail.com> wrote:
In that particular gel I was testing primer dilution so they are the same setup except the second well from the left is 100x dilution then 250x then 500x and 1000x dilution. I was testing to see if my primers were too concentrated.
The left most well is a 1kb ladder
I've also run PCR with plasmid bearing bacteria as the template and not a miniprep with the same results.
I'll keep working of tweaking the procedure to get the ethanol out of my minipreps they usually float away when I try to load them into a well. I've tripled the drying time in the centrifuge, maybe I'll just let the spin column sit in the tube and dry 10 minutes for before eluding.
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