We want to ligate Illumina adapters to already barcoded PCR-products, using a TruSeq PCR-free library kit. Have anyone tried it? Are there any published protocols?
So instead of starting with sheared DNA we will have a couple of different amplicon sizes (~100 bp, ~150 bp). They will be purified with AMpure XP beads and quantified and pooled. Should we then continue with end-repair? I saw elsewhere that this step is needed for for 5' phosphorylation. How much DNA input do we need? The Illumina protocol for shotgun libraries suggest 1 ug (before shearing), which will probably be very difficult to acchieve with our amplicons. Since there will be no two-sided size selection I guess we can use far less, but what is the minimum?
Jon
So instead of starting with sheared DNA we will have a couple of different amplicon sizes (~100 bp, ~150 bp). They will be purified with AMpure XP beads and quantified and pooled. Should we then continue with end-repair? I saw elsewhere that this step is needed for for 5' phosphorylation. How much DNA input do we need? The Illumina protocol for shotgun libraries suggest 1 ug (before shearing), which will probably be very difficult to acchieve with our amplicons. Since there will be no two-sided size selection I guess we can use far less, but what is the minimum?
Jon
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