Amplicon sequencing - fusion primers ordering

CottonJack · 07-05-2013, 12:30 AM

Dear all,

we would like to order fusion primers for 454 GS Junior amplicon sequencing.

When consulting the manual, we are not sure if the primers need to have Phosphorothioated DNA bases on the ends and if B primer needs to be labeled with biotin (or is this true just for shotgun sequencing)?

Thank you for your help.

MEJ · 07-11-2013, 08:05 AM

Hi,

The phosphorothioate bases are there to inhibit any exonuclease activity, so you can order them without. It did work in our hands but if you get any contamination you'll have a problem!

The biotin on the B primer is necessary for the enrichment (enrichment beads are streptavidin coated magnetic beads) of the amplified beads, regardless the type of library.

ajthomas · 07-11-2013, 10:11 AM

For the FLX, the primers do not need to be biotinylated; standard primers work fine. Biotin doesn't come into the process until the enrichment stage, and the biotin there comes from the enrichment primer, not from the primers you used to create your library. Furthermore, only one molecule of the original library you make with your primers will end up on the bead and even if it was biotinylated, the biotin molecule would be stripped off (along with that original molecule) when you add the melt solution.

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07-05-2013, 12:30 AM	#1
CottonJack Member Location: Europe Join Date: Dec 2012 Posts: 11	Amplicon sequencing - fusion primers ordering Dear all, we would like to order fusion primers for 454 GS Junior amplicon sequencing. When consulting the manual, we are not sure if the primers need to have Phosphorothioated DNA bases on the ends and if B primer needs to be labeled with biotin (or is this true just for shotgun sequencing)? Thank you for your help.

07-11-2013, 08:05 AM	#2
MEJ Junior Member Location: Amsterdam Join Date: Dec 2009 Posts: 4	Amplicon sequencing - fusion primers ordering Hi, The phosphorothioate bases are there to inhibit any exonuclease activity, so you can order them without. It did work in our hands but if you get any contamination you'll have a problem! The biotin on the B primer is necessary for the enrichment (enrichment beads are streptavidin coated magnetic beads) of the amplified beads, regardless the type of library.

07-11-2013, 10:11 AM	#3
ajthomas Senior Member Location: Utah Join Date: Mar 2010 Posts: 166	For the FLX, the primers do not need to be biotinylated; standard primers work fine. Biotin doesn't come into the process until the enrichment stage, and the biotin there comes from the enrichment primer, not from the primers you used to create your library. Furthermore, only one molecule of the original library you make with your primers will end up on the bead and even if it was biotinylated, the biotin molecule would be stripped off (along with that original molecule) when you add the melt solution.