Hello,

I've been having some trouble preparing my SOE products for sequencing.

I've been trying to insert a deletion into a bacterial genomic sequence. I used Phusion polymerase to obtain my two homology arms in the first round and to amplify them together in the second one, and the gels for those steps all looked great (except that for the second round, I was also getting faint additional bands of different sizes; there's been a little non-specific amplification; but the only really bright band I was getting was the expected size for my product).

However, when I've tried to run a DreamTaq Green PCR using the final SOE product as template along with my sequencing primers, I haven't gotten any amplification whatsoever; the gel lanes are empty.

I've just had another look over my primers and product (using SnapGene), and everything should be fine, judging from the last Phusion gels. I have absolutely no idea what might be going wrong in between that and the Green gel; could anyone offer a suggestion as to why that might happen? (also, if you know exactly what's going on, please don't just hand it to me; if you'd like, please just offer me a couple of hints, I wanna try to understand what's going wrong, not just get told about it. Cheers!) My only suspicion at this time is that the sequencing primers might be bad, but not that sure about it...

TL;DR: After Phusion PCR to ligate two homology arms with complementary ends, and confirming on a gel that the product is the right size, I get no amplification in a subsequent Green Taq reaction with sequencing primers and I have no idea why

Thank you :D