There is nothing more frustrating than getting back rubbish data from a DNA sequencing run, especially when you are waiting for an important result. For example, confirmation of that clone you have been trying to get for the past three months!
A lot of the time, the quality of sequencing data is within your control. Most DNA sequencing labs run a pretty tight ship, so the source of any problems is often the quality of the template and the primers you provide. The good news is that the chances of success are high with the right template and primers.
Here, we present our top 8 tips for preventing DNA sequencing failure and getting the best results all the time. These tips have been put together with small sequencing projects in mind. However, many of them also apply to bigger applications such as long-read sequencing and long-range sequencing. If you do plan to carry out long-range sequencing, make sure you use a polymerase capable of high fidelity long-range PCR.
1. Don’t Skimp on DNA Extraction
Whether you need to sequence a plasmid, PCR product, or genomic DNA, template quality is critical. Make sure to use good miniprep and genomic DNA extraction protocols to isolate high yield DNA, which is free of E.coli gDNA (if sequencing plasmids), RNA, endotoxins, and salts.
Fortunately, most commercial miniprep kits yield high-quality plasmid DNA for sequencing as long as you follow the protocol and use fresh E.coli cultures. Sequencing is one situation where you don’t want to use a home-made miniprep protocol or recycle your miniprep columns! If you are struggling to get good quality DNA from a miniprep, a midiprep kit may be worth a try. You can find more information on how to make great quality midipreps here.
2. Clean up Your PCR
If your template is a PCR product, make sure that it is clean (i.e., free of PCR reagents) before sending it for sequencing. You can purify PCR products using a range of commercial PCR cleanup kits.
3. Do your own quality control
Whatever your template is, you need to ensure that the final concentration is in line with sequencing requirements and that there is no contamination or impurities. Quality control should be carried out using both spectrophotometry and agarose gels.
Spectrophotometry will reveal the DNA concentration and potential protein or salt contamination. Agarose gels will allow you to detect contaminating gDNA (appearing as a smear throughout the lane) or non-specific bands and can be used to estimate DNA concentration. See this article for a detailed explanation of how to quality control check your DNA.
4. Read the DNA sequencing instructions
Dilute the template DNA to the specified concentration and make sure you send a sufficient volume. This may seem simple and obvious, but not getting this part right is a common pitfall.
5. Use the right primers
Your primers should have the following characteristics:
- A calculated Tm between 50 and 60 °C.
- Minimal secondary structure and potential for primer dimer formation.
- 18-24 nucleotides in length.
- 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.
6. Include a Positive Control
A little extra effort can go a long way! As a control, include a template and primer combination that has been verified to work in a previous sequencing run, as this can help flag situations where any fault is with the sequencing service and not you.
7.Choose a Sequence Provider That Re-Runs Failed Samples for Free
Sometimes sequencing reactions don’t work first time, for no apparent reason, but work fine after a re-run. Most (but not all) providers do re-runs for free, so get maximum value for money by choosing one that does.
8. If Sequencing Doesn’t Work Repeatedly, Try Somewhere Else
Although sequencing services are pretty reliable, poor maintenance of sequencing machines is not unheard of. Find a backup sequence provider that you can quickly turn to if things suddenly go haywire at your normal provider.
So they were our top tips! Do you have any more to add?
Originally published in 2009, republished in 2017.