Consistency is Key: Creating beautiful Sequences every time
When you work with DNA Sequencing long enough, you begin to look at chromatograms like they are works of art. When I see a chromatogram with high, defined peaks, a flat and clean baseline, and a sequence that seemingly goes on forever, I consider it “beautiful.” In a stark contrast, when the primer fails to anneal to a template completely, you get nothing but N bases, the background noise is indistinguishable from the peaks, and everything looks chaotic. To me, this looks like a train wreck, and my brain starts working to figure out why it doesn’t look “beautiful” and how to re-do it to make sure that it does.
The Beauty
and the train wreck:
With the combined years of experience everyone at Eton has, we can all agree on one thing: consistency is the key to having a “beautiful” sequence every time. Order your oligos from the same company, treat them with the same process, and we can help you troubleshoot your way to get your orders right every time.
One great example is a customer we had that had consistently high concentrations and when we diluted to the normal amount, we could not get it to the data that they required. So we set up a troubleshooting experiment to look at the sample with a few different concentrations, and we found one that worked the best with their samples. Now we use that protocol every time and they get great results consistently.
We had a customer who had beautiful results with our list of universal primers, but with their custom primers,
the data was inconsistent. We asked for the sequence, made one from the company we use for oligos, and re-ran it. It worked. So we found out that they were using a poor quality oligo. We were able to deliver to them the oligo we used, and now they use it on all their samples, so the data is consistent for them every time.
We test every order with a nano-drop spec, but we do recommend running your samples (especially PCR products) on a gel to confirm the concentration. There can be discrepancies from the nano-drop (due to proteins or RNA being in the sample) so confirming it by gel is more accurate with PCR products. Which will keep your results more consistent.
So, in short, if you treat your samples the same and they have a similar 260/280 number as well as a similar concentration number, you will likely get your results to look great the first time. If not, we will be able to work with you and figure out what the problem is.
If you ever have any questions about this feel free to call our customer service in San Diego at 800.758.1630 or our customer service in North Carolina at 919.314.5539. They will be more than happy to help you with troubleshooting DNA sequencing .
