you will get no sequence under the primer or for the next 30 bases with Sanger sequencing so 2 possibilities are cloning the fragment and then using sequencing primers 60 bases into the plasmid away from the amplimer or use pyrosequencing which gives you all sequence from the primer but is expensive . Theoretically you could add more dideoxy bases to the sequencing mix to get sequence close to the primer but that would be a lot of work and cloning works.

Marcela Krutova m13 tailing is a good suggestion that works well and also means that any amplimer can be sequenced with the same sequencing primers but I think that the sequence close to each primer end would probably only be cleanly sequenced from the other end of the amplimer

Dear Andrew Jenkins

Sometimes the easy solution is in this case to use a lower PCR product input into the Sequencing reaction. You can also try to use M13 tailed primers.

Good luck,

Marcela

you will get no sequence under the primer or for the next 30 bases with Sanger sequencing so 2 possibilities are cloning the fragment and then using sequencing primers 60 bases into the plasmid away from the amplimer or use pyrosequencing which gives you all sequence from the primer but is expensive . Theoretically you could add more dideoxy bases to the sequencing mix to get sequence close to the primer but that would be a lot of work and cloning works.

Marcela Krutova m13 tailing is a good suggestion that works well and also means that any amplimer can be sequenced with the same sequencing primers but I think that the sequence close to each primer end would probably only be cleanly sequenced from the other end of the amplimer

Thanks Paul, Marcella,

I'll look into the M13 tailing trick. Even if it just gets us another 20 bp each side it will make a big difference.

for short sequences you may not gain much as the sequence close to the primer will be well sequenced from the other end if you are sequencing in both directions. Sequencing kits are designed to sequence long sequences because that is what everybody wants most of the time. In your case most of the sequencing kit is wasted so you might try misusing the kit. Sequence much 5 times as much template as the kit recommends and use much more sequencing primer than recommended. The kit will run out at shorter sequences but that is what you want and the extra primer amount will generate sequence nearer to the sequencing primer

Dear Andrew Jenkins,

You can try Bigdye V1.1(with more ddNTPs) instead of V3.1, which is more compatible with short PCR products sequencing.

When using V1.1,the PCR product shows 100% basecalling accuracy beginning with the first base adjacent to the primer :

https://assets.thermofisher.com/TFS-Assets/LSG/brochures/cms_040026.pdf

hope this link is useful

seqanswers.com › ... › Sanger/Dye Terminator

regards