Homemade TruSeq adapters

[sebbaba]

Hi all, for various reason I am generating homemade TruSeq adapters. After annealling the universal and index oligos, they run at ~65bp on a 2.5% gel as I expected. However, Illumina TruSeq adapters from the kit, run as a positive control, were at ~100bp. Anyone have any experience of why this is (given they are supposed to be ~65bp)? Is it anything to do with the Y structure and does this, presumably, mean that my homemade adapters have not annealed properly?

Thanks,

nb. To anneal I mixed eqimolar amounts of index and univeral oligos in 50mM NaCL, incubated at 95C for 5 min and allowed to cool to RT in the heat block (2hr). The oligos are modifed 5'phosphate and 3'phosphorothioate, respectively.

[SeqR&D]

Are your adapters methylated? Do you know that you are using the same sequences with the same double stranded/single stranded lengths? What do they look like on a denaturing gel?

[NextGenSeq]

Also, you have to have them synthesized with a phosphothioate bond between the last two 3' bases.