sequencing with specific primers

klytie · 08-04-2011, 03:11 AM

what kind of conditions should one bear in mind when designing custom sequencing primers (instead of using Illumina universal sequencing primers).
Also, is it possible to ligate or add flow cell adaptors by PCR directly to the insert and then use the nucleotide sequence of the adapter to start the sequencing?

I would very much like to skip the ligation+PCR with Illumina oligos but the thing is, I only have information on one end of the insert (app 300bp) and I need to do the multiplex (by adding index to the other end because otherwise it would be too far from the important information - app 20nt). Most probably it will be a paired end sequencing.

Help...

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08-04-2011, 03:11 AM	#1
klytie Junior Member Location: Tallinn Join Date: Jul 2011 Posts: 5	sequencing with specific primers what kind of conditions should one bear in mind when designing custom sequencing primers (instead of using Illumina universal sequencing primers). Also, is it possible to ligate or add flow cell adaptors by PCR directly to the insert and then use the nucleotide sequence of the adapter to start the sequencing? I would very much like to skip the ligation+PCR with Illumina oligos but the thing is, I only have information on one end of the insert (app 300bp) and I need to do the multiplex (by adding index to the other end because otherwise it would be too far from the important information - app 20nt). Most probably it will be a paired end sequencing. Help...