The peaks here are usually unresolved and small, so I suggest designing your primer at least 50bp upstream of the sequence of interest. Never trust the first 20-30 bases of a DNA sequencing read Anything more and you’re venturing into the uncertain terrain. Expect to get 500-700 bases of clean reliable DNA sequenceĪnything less and you might suspect contamination in your sample or consider asking your sequencing facility to apply a special protocol for a difficult template. You should see individual, sharp and evenly spaced peaksģ. You can use any of the following programs to view your. Guidelines to help with DNA sequencing troubleshooting and analysis 1. In fact this is so ambiguous that the DNA sequencing reaction should be repeated.
If you never looked at the trace you would be happy.īut look closer, the overlapping peaks in the chromatogram suggest the results are not as certain as the sequence may suggest. Chromatograms come to the rescue for analyzing and troubleshooting your DNA sequencing resultsĪbove (top right corner of this post) is an example of a seemingly clean DNA sequence (no Ns in sight). And, like all controls, missing out is a big mistake. When it comes to DNA sequencing the chromatogram is your visual control. These controls help you properly visualize your results.The most important of those is to always look closely at the trace file (or chromatogram) of the sequencing results you get back from your favorite sequencing facility. When you run a restriction digest on a gel you always include proper controls like uncut DNA and the proper ladder. So I have developed some good habits that I wanted to pass on to you to make sure you are getting the most out of the data you get back from your sequencing runs. This blog was originally published on BitesizeBio here.Īs part of my job ensuring plasmid quality at Addgene, I analyze 50-100 sequencing reactions a week.
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