Re: [DIYbio] Re: PCR trouble

The primers bind correctly to the appropriate regions.

A few things:
You should get some NEB 2-log ladder to replace the ladder you are using. It has bands down to 100 bp and will make interpretation much easier.
Also, you can run your gels much longer.

Your problem is likely one of two things:
1) Far too much template. This is a common error. PCR can amplify (in principle) a single molecule. You need vanishingly small amounts of template. Plasmid DNA preps often contain many inhibitors of PCR reactions such as ethanol or SDS. I'd strongly suggest doing serial dilutions of your template DNA down to 1:1000 and adding 1 ul of the result as template. This eliminates most of the inhibitors and leaves sufficient template. Or, you can do colony PCR with cells. Barely touch a colony with a fine tip then swirl the tip in 50 ul of water, scraping it on the tube edge. Change tips, use 1 ul of this as a template. Your initial denaturing should be sufficient to lyse the cells. Remember — less is more.

2) Your extension may still not be long enough. Pure Taq has problems with long templates. I'd definitely switch to Q5. I would get the master mix and not fool with separate buffer/enzyme/magnesium. The manufacturers know what they are doing in making these master mixes, and it is one fewer thing to screw up in pipetting. Q5 will allow a much shorter extension time. Don't forget to follow instructions on the cycling conditions, which are quite a bit different from Taq.

> On Mar 2, 2016, at 12:

> 30 AM, David Ishee <midgardkennels@gmail.com> wrote:
>
> I designed them myself, my first try at primer design, so it's very possible that it's a design issue.
>
> I'll get my mini preps to a clean enough point to run one with a digest as well.
>
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