GS Junior - amplicon primers

[mikebravo]

Hi all,

we just got our GS Junior and I am currently designing primers for amplicon sequencing. Does anyone have some experience with it? For example which amplicon size is acceptable for biderectional sequencing? Is there a cut of in primer length an GC content? What is the maximum acceptable size difference for amplicons in emPCR not affecting the coverage? I know that Roche gives some parameters but im really interested to know what will work in real life.

Thank You all!

[junioshi]

Hi mikebravo,

Hi,

if you wish to do bidirectional sequencing an amplicon length of about 400 bp is acceptable, i think. In our training course we were advised to minimize the size differences to a maximal range of 100 bp. We use amplicons with a length ranging from 370 to 470 bp. In general all requirements that are essential for all-day primer design have to be taken into consideration when designing your NGS-Primer. The length of my primers range between 50 and 58 bp (with MID, Key and A or B). I wish you good luck for your experiments with your GS Junior.

Greetings from Berlin