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-   -   Nextera XT vs TruSeq PCR free (http://seqanswers.com/forums/showthread.php?t=44033)

vl80 06-08-2014 11:06 PM

Nextera XT vs TruSeq PCR free
 
I have previously used the TruSeq DNA kit (only once) and now need to move on to use either Nextera XT or TruSeq PCR free kits and perform the whole library prep alone for the first time.
I would greatly welcome opinions on which of the two kits have given better results for small genomes (bacterial)

2)
For Nextera XT, is it better not to use the bread normalization and do the traditional quantifications? There seem to be mixed experiences for using bead normalizations.

3) is there anyone using Qubit alone for library quantification? Or is it better to use KAPA qPCR instead or both?

4) This is probably a very naive question,
for Nextera XT with bacterial genomes, is it necessary to sonicate/ nebulize to get a larger size fragment and then use the kit based tagmentation than the kit alone.

Thanks a lot

nucacidhunter 06-09-2014 01:13 AM

1) For bacterial genome it would not make much difference in result. The consideration would be cost and availability of DNA input amount.

2) qPCR based quantification (not using normaliser beads) will increase chance of even number of read output for samples.

3) KAPA qPCR certainly is better than Qubit. Qubit only measures mass of DNA not cluster ready portion of library.

4) This question is a bit unclear to me. Using high molecular weight input DNA (without sonication) will give more consistent results. Sonication is not in the protocol and I do not see any reason for sonication.


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