I have a question about library preparation using NEBNext multiplex oligo for Illumina (index primers set 1). During PCR enrichment of adaptor ligated DNA, both the universal PCR primer and the index primer are added. I have noted that the complimentary regions (to the NEBNext adaptor for Illumina) of both these primers is identical (5'... GCTCTTCCGATC*T 3') and for both is at the 3' end. Therefore during PCR 3 types of products will form; the target DNA with the universal primer at both ends, the target DNA with the index primer at both ends and the target DNA with the universal primer at one end and the index primer at the other. This doesn't seem logical to me as some product will not have the addition of the index. I think therefore I must have made a mistake but I cannot understand where. Can anyone help me in letting me know how the index primer and the universal primer bind to the adaptor ligated DNA and thus what product will be formed. Thank you.
Edited by Rje, 06 December 2013 - 05:31 AM.