Hi all,
I'm working with analyzing DNA taken from prey. My goal is to find a predator who killed. I'm isolating DNA from swabs (saliva), with GeneALL micro DNA kit, then I'm making PCR with D-loop markers specific for canidae. PCR is cleaned with FastAp. Final product is send to company for sequencing.
And sometimes I'm struggling with outcome like on attached picture. Did anybody know what I can change? Should I dilute my PCR before sequencing? Or maybe there is some problems with cleaning PCR or isolate?
Any help will be welcome.
Can you attach an original .abi sequence file please?. These files do contain useful information. I agree with Dirk Schmidt
about the cleanness of template dna for sequencing and thast ,along with not removing the pcr primers, is the usual reason for mixed sequences
I agree with Paul Rutland, you didn't remove correctly the primers from your PCR before sequencing
Thank you all very much for answering.
Unfortunately on gel there is one fat, light line of DNA. I assume, that this is same species that killed.
Paul Rutland I'm attaching .abi Can you show me how and what informations you can get from .abi file?
Guy Longepied how can I remove primers before sequencing? I assume there is some kit for that, can you recommend best one? For cleaning PCR products I'm using FastAP, but it looks like it didn't work. Moreover, I asked sequencing company for cleaning one of my PCR product before sequencing, to check if I have problem with FastAP, and the outcome was this same as from cleaned by me samples.
I understand that your problem is not cleaning PCR products. You have a complex
mixure. For me to understand what happen you can clone your PCR product with a TA cloning vector and then amplify the inserts directly from bactéria with vector primers and do sequences forward and reverse.
There is certainly another solution with your primers in your D-loop markers
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