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Old 02-17-2014, 06:19 PM   #1
Location: Perth

Join Date: Sep 2012
Posts: 55
Default Short LNA custom sequencing primer for MiSeq

Hi All,

For a couple of reasons we are trying to 'shorten' the custom sequencing primer we use in MiSeq. Primarily it is because we are trying to generate 'complete' fusion-primers in a single step. I have two queries that I was hoping to get comment on.

1) Has anyone used a LNA primer shorter than the 22bp fluidigm primer (A+CA+CTG+ACGACATGGTTCTACA). I am tempted to add a few more LNA's and remove some 3' bases but was hoping someone might have done this already?

2) We tried (very unsuccessfully) to overlap the sequencing primer into the adaptor(s) - has anyone managed to achieve this? The fluidgm LNA primer overlaps by 4bp into the P5 adapter.... but i was hoping that someone in SeqAnswers might have cracked this.

The upshot is that we feel we have lost PCR efficiency (assessed by qPCR) when compared to fusion-tagged 454 and PGM adaptors (which do not have separate sequencing primers). Any thoughts/suggestions would be gratefully appreciated. Best, Mike.

Last edited by bunce; 02-17-2014 at 09:14 PM.
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Old 02-24-2014, 02:14 AM   #2
Location: London

Join Date: Nov 2012
Posts: 96

I know that some people on this forum just add some padding nucleotides before the adapter, so you can extend the sequencing primer back into that, bringing your Tm up. I was certainly once told by an Illumina rep that they'd be wary of engineering overlap between the lawn oligos and sequencing primers.
JamieHeather is offline   Reply With Quote

custom primers, fusion primers, lna, miseq

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