How to design the primer for Sanger sequencing? I'd like to know the sequence of a 2kb inserts.
Is there anything special that should be noticed? From primer to the interested sequence, how many nt should there be? Also, usually what's the max length for normal Sanger sequencing?
Is there any good online tool or software that can help to design the sequencing primer?
Hello,
Usually a good length is 15-20 nt. The main thing you want to avoid for a sequencing primer is strong self-complementarity, either as a hairpin or homodimer. You can check this using the website https://www.idtdna.com/calc/analyzer. If your primers do not work, checking these parameters could explain why. Honestly, most of the time they just work in my experience.
You can also use Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). You input your full 2 kb and it will design good primers (in regards to Tm, weak hairpins/dimerisation) for you. Change the "Task" dropdown to sequencing. I did this once and the primers worked well.
You can normally get ~800 bp of good quality sequence, so you will need to order multiple primers to sequence the full 2 kb, maybe 5-6 depending on your DNA quality.
Good luck!
Hello,
Usually a good length is 15-20 nt. The main thing you want to avoid for a sequencing primer is strong self-complementarity, either as a hairpin or homodimer. You can check this using the website https://www.idtdna.com/calc/analyzer. If your primers do not work, checking these parameters could explain why. Honestly, most of the time they just work in my experience.
You can also use Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). You input your full 2 kb and it will design good primers (in regards to Tm, weak hairpins/dimerisation) for you. Change the "Task" dropdown to sequencing. I did this once and the primers worked well.
You can normally get ~800 bp of good quality sequence, so you will need to order multiple primers to sequence the full 2 kb, maybe 5-6 depending on your DNA quality.
Good luck!
There isn't much I can add to Rory's excellent answer. I will therefore simply attach an SOP I made a while back detailing good primer design (including for Sanger sequencing) which expands on seminal points made already by rory
Hi,
The excellent answers provided by Rory Little and Laurence Stuart Dawkins-Hall leave little room for additions. I liked a lot the SOP by Laurence Stuart Dawkins-Hall and I would like to know if it is OK to distribute (with proper attribution of course). Have you published it anywhere?
Cheers,
Anibal
Dear Anibal. Yes of course you can distribute my SOP. No it hasn't been published
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