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Old 07-23-2014, 02:43 AM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,232
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Could I size select the samples to remove the smaller degraded fragments?
Smaller fragments will be cleaned after shearing and also after end repair, so an extra size selection will not make much difference.

I had some trouble interpreting your gel run. The agarose concentration and run voltage are too high for genomic DNA resolution. I am not sure if some DNA has stocked in the wells or not and it seems there is no DNA in some lanes.

The best that one can do with degraded DNA is to repair it first using DNA repair enzyme mix. Shearing condition in Illumina user guide is for high molecular weight DNA, so you might need to optimise shearing condition for your typical degraded sample to achieve fragment size distribution similar to recommended input DNA. Input DNA also can be increased to 2x to compensate for low quality and fragments that will not contribute to library because of excess damage.

Library can be prepared with Nano kit using even pg amount of DNA. The downside of preparing a library from very low amount and degraded DNA is that the library may not have high diversity or representation of some region might be affected. Sometimes these effects can be tolerated depending on the aim of experiment.
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