Whether you’re new to Sanger sequencing or a regular, researchers are often trying to use Sanger sequencing as a ways to confirm their target DNA sequences. Prior DNA handling may have an effect on downstream Sanger sequencing, giving you poor read quality. No one likes to deal with messy Sanger sequencing, so we’ve put together 5 tips to help you design and run your Sanger experiment, even if you are using Sanger to confirm your NGS variants or for your CRISPR/cas9 gene editing workflows.
Our SeqitOut video series tackles all things sequencing (from Sanger to NGS). To help you get started on your Sanger experiments we’ve put together five tools to help you kickstart those projects. And if a question pops into your head while you’re watching the videos, just send them in at thermofisher.com/ask – the team at Thermo Fisher Scientific just might make an episode out of it!
So let’s get you started….
The most commonly asked question by researchers is trying to understand if Sanger sequencing is sufficient for their experiments, if they should be using Next-generation sequencing or both, one for discovery and another for orthogonal confirmation. Understanding these methods early on, will help you plan and design your experiments accordingly. If NGS is better suited for your experiment – check out our 5 Things to Help Start Your NGS Experiments blog.
So you decided you want to use Sanger sequencing, but what exactly is Sanger sequencing and how does it truly work? Here is a quick recap of this widely used method, where you’ll learn about the basics of Sanger sequencing.
Alright you have your precious DNA samples ready, you’ve run your PCR, but what are the best ways to clean your DNA before Sanger Sequencing? Correctly cleaning up your sequencing reactions is an integral part of the Sanger Sequencing workflow. Without cleanup, you will get suboptimal sequence data. To understand the importance of sequencing clean-up, you first have to understand the BigDye Terminator sequencing workflow and the basics of how the sequence information is captured. In short, the DNA that you want to sequence should be isolated and purified. This can be done by a variety of methods. But in general, the cleaner the product, the better the results.
You’ve properly cleaned your PCR and are ready for sequencing, but how do you set up your run? Sanger Sequencing has moved from a very manual process using gels to being automated with capillary electrophoresis. As with all technologies, Sanger Sequencing has gotten easier through the years and we want to share with you just how simple it is to set up a Sanger Sequencing run. Watch the video below as we walk you through how to set up a Sanger sequencing run. If you’ve run Sanger sequencing before, but have messy Sanger reads near the primers – check out our blog for ways to help resolve this issue.
You’ve used NGS for your experiments and want to confirm your targets using an orthogonal method. Sanger sequencing is often used to confirm NGS variants, but we’ve got you covered. Here is a simple workflow to help confirm your NGS variants.
Have more questions? We’ll help you get them answered! Submit your questions at thermofisher.com/ask
For more information:
Learn about CRISPR-Cas9 mediated genome editing workflow with Sanger Sequencing here.
Check out our complete guide to Sanger sequencing from Education to Application here.
Learn more about our Sequencing solutions here