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Old 11-13-2014, 12:08 PM   #1
esherman
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Location: USA

Join Date: Jul 2013
Posts: 14
Default 'Assembly PCR' for homemade amplicon library prep

I'm trying to make homemade amplicon library preps for the Illumina MiSeq, and my fusion primers always end up being around 100bp (if not more!) by the time I add all the necessary sequences (p5/p7, indices, primer landing sites, N's for diversity, etc.). This requires I buy oligos at the 250nm scale of synthesis rather than the standard 25nm scale. When I'm buying a whole plate of 96 indices, this gets very expensive very fast!

I'm thinking of an 'assembly PCR' approach in which I order four 25nm oligos. Two short ones that contain the sequence-specific sequence fused to the Illumina sequencing primer landing sites and two additional short ones that contain the p5/p7 sequences, indices, and a bit of the primer landing site. These could all be thrown into a single PCR reaction (perhaps with a bias towards the 'outer' primers containing the p5/p7 sequence).

Of course I would have to pay attention to strandedness and directionality when designing the oligos, but I wanted to see if anyone has tried this before I spend too much time thinking about it.

This idea would also have the benefit of being able to change out the sequence-specific 'inner' primer and re-use the outer primer/indices for different experiments.
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