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Old 06-04-2013, 05:49 AM   #3
Location: Nottingham

Join Date: Jul 2012
Posts: 29

Hi Phillip,

I haven't denatured the adaptors, I had read in a troubleshooting guide that a 70 hold was needed to ensure no secondary artefacts are present. I had miss understood and this is actually part of the thermocycler ligation program. So no damage done

Illumina have replaced the kit however so I have fresh reagents.

The new PCR free kit comes with a Ligation Mix which is kept at -20, the adaptors were handled following the protocol, which states RT thaw. They were then used straight away, but will now try thawing on ice.

I also suspect a contaminant in the DNA, and as or DNA is normally prepared in to TE buffer I have done a phenol DNA prep and resuspended an ethonol washed DNA pellet in the Illumina buffer with RNAase A, fragmented, ampure xp clean up and resuspended in Illumina RSB buffer, but it still failled.

I'll have a look into the Zymo kit thanks. Would you do this and the RNAse step post fragment?

Thanks for the input
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