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Old 05-28-2013, 05:52 AM   #1
Location: Nottingham

Join Date: Jul 2012
Posts: 29
Unhappy TruSeq PCR Free LT Kit Issues

Hi all would appreciate any input on an issue i'm having.

I've moved across to the TruSeq PCR free kit and seem to be struggling with generating a library.

I'm fragmenting by covaris to 550bp in 50ul RSB as described and I've followed the rest of the protocol to the letter, but my best prep is still only 14pM of ligated adaptors (according to Kapa qPCR quantification). I still have approx 400ng of DNA left (quantified by qubit) which i was expecting due to the size selection steps using the beads.

I've asked tech support and they aren't much help.

I've read that sometimes the adaptors need to be denatured before ligation, Illumina replied with "the protocol has been optimised for the conditions published".

Any ideas would be much appreciated

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