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-   -   Best method to prepare 100-200 bp DNA sample (Micrococcal Nuclease Digested) (http://seqanswers.com/forums/showthread.php?t=33811)

spf2 09-17-2013 01:30 PM

Best method to prepare 100-200 bp DNA sample (Micrococcal Nuclease Digested)
 
Hi Everyone,

We're trying to map the nucleosome occupancy of a yeast. At the moment, I have micrococcal nuclease digested DNA fragments. These are around 100-200 bp long and I was wondering what kit or preparation would be best to prepare them for sequencing on the Illumina HiSeq system. Our lab has tried the Nextera XT kit, however this did not work because of the input DNA was less than 300 bp long. I was wondering if anyone knows anything about the TruSeq Nano DNA sample preparation kit or the TruSeq ChIP sample preparation kit and whether or not they would work for small fragmented DNA such as this?

Spf2

kcchan 09-17-2013 05:24 PM

The TruSeq DNA kits should work fine with DNA fragments of that size assuming you have at least 50-100ng of DNA available.

rnaeye 10-11-2013 09:07 PM

I have been using NEB's NEBNext® Ultra™ DNA Library Prep Kit for Illumina. You can make library as little as 5 ng human DNA. I am sure with yeast DNA you can even use lower input amount. Hope helps.

spf2 05-14-2014 12:25 PM

I used NEB's NEBNext® Ultra™ DNA Library Prep Kit for Illumina to generate nucleosome libraries for the yeast. They looked great on the bio analyzer/qubit, but over clustered on the miseq resulting in unindexed data with all of it being undetermined. I was wondering if you have any suggestions for optimizing the libraries for Illumina or any idea what may have went wrong?

nucacidhunter 05-14-2014 01:22 PM

Overclustering occures when too much library DNA is loaded into flowcell. If the libraries do not show adapter or primer dimers on bioanalyser trace, sequencing can be repeated with less input. Overclustering is an tindication of poor quantification.


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