Purification of Nextera libraries for use on the MiSeq

[vieiraL]

Hi everyone,

I am about to start preparing a nextera library to run on the MiSeq. The only thing I don´t available right now is the AMPure kit for library purification. If the purpose of this kit is to remove free nucleotides and small fragments from the library, can I also achieve the same goal with column-based PCR purification kits? If so, which are the best kits to use?

Thanks in advance.

[kcchan]

You can use any column based PCR purification kit in place of the Ampure XP beads. The old Epicentre kits use Zymo DNA Clean & Concentrator 5 or Qiagen MinElute kits with the standard protocol, but any similar kit should work fine.

[RCJK]

If running a 2x250bp run, you'll also want to get rid of some of the smaller fragments. That's why they tell you to use less beads for the longer runs.

[lorendarith]

Depending on the size of your mate pair library MinElute may not be suitable til you fragment it for sequencing (TruSeq protocol).

[ramujana]

Hello,
i face the similar problem what to use instead of Ampure (i'm dealing with 2x150bp reads on MiSeq, Nextera XT DNA prep and pooling amplicons). However, on the BC website i find information comparing Ampure, qiaquick, and quickstep, showing that ampure gives better yield.
Does anyone has experience using Zymo DNA Clean or ThermoFisher (Fermentas) GeneJet cleanup? What would be critical points to consider while choosing particular alternative? Thanks,

[ramujana]

BTW, i could also argue that simple Sephadex G50 filled collums would also work for clean-up - any commens on this?