About the author

Nick Oswald

Nick is a molecular biologist-turned-publisher. After a PhD in Developmental Biology and an eclectic seven years in biotech he is now Editorial Manager of Neuroendocrinology and the founder and Editor-In-Chief of Bitesize Bio. You are welcome to connect with Nick on LinkedIn

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There’s nothing more frustrating that getting back junk data from a DNA sequencing  run. Especially when you are waiting for an important result, like confirmation of that clone you have been trying to get for the past three months.

Most (but not all) of the time, the quality of the results you get back from the sequencing lab are within your control. Most (but not all!!) sequencing labs are pretty well set up so the source of any problems is normally the quality of the template and primers you provide.

The good news in this, of course, is that if you follow the procedures to make sure your template and primers are just right, then you’ve for a pretty good chance of success.

If you are struck with junk DNA sequencing results, it helps to have a checklist to work from.

So so here are my 8 top tips for preventing DNA sequencing failure and getting the best results all of the time.

1. Don’t skimp on the miniprep. Quality of template is critical – plasmids for sequencing must be of a reasonable concentration and free of E.coli gDNA, RNA, endotoxins and salts.

Happily, most commercial miniprep kits give good enough quality plasmid DNAdoe  sequencing if you follow the protocol properly and use a fresh E.coli culture. So this is one situation where you don’t want to use a home-made miniprep or recycle your miniprep columns

If you are struggling to get good quality DNA from a miniprep, a midiprep often gives better results and is worth a try. For more on how to make great quality midipreps, click here.

2. Clean up your PCR. If your template is a .PCR product, you also have to make sure that it is nice and clean before giving it to the sequencing lab so make your you do a PCR purification using a good quality kit.

3. Do your own quality control. Whatever your template is, you need to check it before sending it to endure that the concentration is high enough and there is no contamination.

This should be done using both a spec and a gel.

The spec will tell you concentration and give an idea of whether there is any protein or salt contamination. The gel will show up contaminating gDNA or non-specific bands and confirm the concentration measured by the spec.

For a fuller explanation of how to quality control check your DNA, click here.

4. Read the sequencing service’s instructions. You need to know the concentration they want the DNA at and the amount of DNA they want. Dilute the DNA to the specified concentration and make sure you send enough.

Simple and obvious, of course. But a lot of people screw up at this easiest of steps.

5. Get the primers right. Your primers should have the following characteristics:

• A calculated Tm between 50 and 60 °C.
• Minimal secondary structure and potential to self-hybridize.
• Length of between 18-24 nucleotides.
• Diluted in dH2O to the concentration specified by the sequencing service.

Primers can be checked using primer analysis software. Our Molecular Biologist’s Toolbar has handy links to a couple of primer analysis tools (and tons of other useful stuff… click here to check it out)

6. Include a positive control with your sample(s). As is so often the case, a little extra effort in setting up a good control can save you a lot of time in the future.

If you have a known good template and primer combination (that has been verified in a previous sequencing run) then you should include it with every sample set you send for sequencing as this can flag up situations where the fault is with the sequencing service and not you.

7. Choose a sequence provider that routinely re-runs failed samples for free. Sometimes sequencing reactions don’t work first time, for no apparent reason, but will work after a re-run. Most sequence providers will do a re-run for free but some won’t so make sure you are getting maximum value for money by going with one that does.

8. If it doesn’t work repeatedly, try somewhere else. As I said above sequencing services are pretty reliable. Relatively recently I had some big problems with poor sequencing data and the problem turned out to be poor maintenance of the sequencing machine at the service I was using. A quick switch to another service provider sorted things out. So it’s a good idea to have a backup sequence provider that you can quickly turn to if you start to get suspicious about results from your normal provider.

So those are my tips.

Do you have anything to add? And what are your experiences of DNA sequencing?