Sanger sequencing


Quality, speed or price - what do you prefer in Sanger sequencing?

Just as few people go to the theater or a sports match without knowing what is on the program, it is normal for any other service you intend to order to have at least some idea in advance of what you can expect from the service provider, especially in terms of QUALITY, SPEED and PRICE. This of course also applies to DNA sequencing. It is understandable that most clients would like all of these three criteria to be met at 100% at the same time, but we do say that this is not basically possible.

Low template concentration – What can you do?

For successful sequencing analysis it is crucial to use optimal template concentration (as described in our guidelines for sample preparation). If you do not have required amount of template, the total volume of a sample can be lowered but the template to primer ratio must be kept. The problem of low template concentration cannot definitely be solved by only increasing the total sample volume...

Higher-quality sequence data at the beginning of electropherogram - Modified sequencing primers

Routinely obtained sequence electropherograms typically start with unreliable (unreadable) data just at the beginning of a sequence. Although we should theoretically read the first base after the sequencing primer, there are often errors or truncations just behind the primer ...

How do I sequence large templates directly?

Here and there we do receive a request to sequence large templates (i.e. chromosomal DNA, BACs, cosmids) directly. We can do this but similarly as for standard samples where short pcr products or plasmids serve as templates also here the key prerequisite is the template amount.

How do you work with .ab1 files?

Results of Sanger sequencing are provided in three types of file formats and not every user of our services is familiar with efficient usage of these data files. Here are brief instructions how to use them properly.

HairpinSeq – a sequencing protocol for difficult templates

Have you ever ordered sequencing analysis of a plasmid or PCR product and as a result received the DNA sequence that suddenly stops? One of the possible causes can be that in your template complex secondary structures are being formed, most often hairpins.

10 REASONS to Sequence at SEQme

Are you wondering why you should send samples to us? Please read our 10 good reasons to answer this question!

A Few Thoughts on Troubleshooting of DNA Sequencing - Part III

Assuming we have good signals and read length is as expected which means we have successfully solved all issues mentioned in previous two parts of this post, we can still be far away from seeing nice data. This third part deals with the problem of having peaks overlapping other peaks. The sequence is not readable.

A Few Thoughts on Troubleshooting of DNA Sequencing - Part II

In the first part we discussed how to troubleshoot no or low signals. Basically empty electropherograms. Very frustrating. Now, I focus on another frequent result – you obtain some signals but the height of the DNA sequencing peaks diminished rapidly. A read length is very short.

A Few Thoughts on Troubleshooting of DNA Sequencing - Part I

It is not the aim of this post to provide a detailed description of all problems you may observe when evaluating your sequencing results. Instead, I focus on most frequent problems and recommend some steps to take for your consideration. Additionally, I am not covering issues related to instruments and sequencing reagents used in our (or any other) sequencing lab because first it is our responsibility to secure a problem-free sample processing on the instrument and second, from the user‘s point of view, it is of low interest because you cannot influence it anyway.

Are you contaminating your DNA on cleanup columns?

A very frequent scenario when cleaning up your samples (plasmids or PCR products) for DNA sequencing is to load your DNA to silica-based membrane in spin columns. Unfortunately some columns (even those of established manufacturers) may not have a really favorable shape and consequently your DNA gets dirty during purification.

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