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cloning with nested pcr - (Dec/01/2011 )

So I'm trying to clone a number of genes from a ds cDNA pool using degenerate primers designed from protein sequence. So the degeneracy is low, and my ds cDNA pool seems good. I've got three sets of primers: F1/R1, F2/R2, F3/R3, with F3/R3 being the most internal. I'm using hifi polymerase and a high GC kapa buffer (it tested better with first product synthesis than the hifi buffer), 25 microliter rxs with: 1 mL template, 0.75 each primer and dntps, 5 buffer, 0.25 polymerase, and the rest water. I ran a temp series 56-68 and the 66-68 range is a better product.

The problem I'm having is that in my controls when I run the second primer set, the internal primers either F2/R2 or F3/R3 with the first round purified produce, one of the primers always produces a band around the same size by itself. I'm just not sure if it's significant. Is it going to cause a big problem? I'm gel-extracting to do the A-tail so I can ligate, but the band I'm talking about is around the same size so it's hard to seperate them (assuming it's even a problem). So for one I was just wondering what to do about this.

The second thing I'm wondering is when I ran the F3/R3 primer set with the 1st round product I got the band on one of my single primer controls, but I didn't get anything for my actual rx. Nothing but a big band of what looked like primers down at the bottom. This was the case for two different species I ran. So that's a little confusing to me.


If I understand what you mean by "by itself" as a negative control, it could be either dimers or contamination (which has increased occurence in nested PCR).
If your product is smaller than about 150bp and diverse from your product it could be dimers. And those can either be present also in your reaction and in that case you will have dimers in your clones too, or they can be template specific and don't create the nonspecific product in the presence of desired amplicon.

If it's a bigger band and is exactly the same size as your product, then contamination is more likely. You can try using everything new, filtered tips, cleaned pipettes, new batch of everything or set the reaction somewhere else than you did now, free of contamination, and see it the band is still there.
Finally, you can try to clone and sequence it to see what it actually is if you eliminate all source of contamination and it's still there.


it's too big, ~500bp, almost as big as the fragment I'm trying to amplify, so I suppose it would be the contamination option. But the negative control w/o template showed no bands, so I at least know it's not contamination in the primer itself. And frustratingly, it doesn't show up in the actual rx with both primers and the template (nothing did), so it's not in the template. The frustrating thing is it's consistent. The same primer by itself always produces a band. Though I suppose the fact that I didn't get the same band in the actual rx with both primers says a lot-- HOWEVER, there I do see a strong band that is dimerization.


Now I don't really follow.
" The same primer by itself always produces a band." "But the negative control w/o template showed no bands."
What exactly is composition of this "by itself" reaction, when you say It's not a negative control?

Also "amost as big" is IMHO not a contamination, that would be "the same".