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Old 02-02-2015, 01:30 PM   #1
libro.s
Junior Member
 
Location: boston

Join Date: Jun 2010
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Default DNA degradation and size selection

Hi,

I used the Covaris to shear gDNA for Illumina libraries, but something went wrong because instead of the expected 500 bp fragments I got extensive (and pretty random) degradation. I am attaching the results from the bioanalyzer: sample 4 is a + control and looked fine, but I had to manually set high and low marker peaks for the remaining 3 samples. S1 and S2, in particular look really degraded. However, they seem to have enough 200-300 bp fragments that might be recovered with a size selection. Has anyone ever successfully built and sequenced illumina libraries from similarly over-fragmented gDNA?

Thank you in advance!
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File Type: pdf HS_DNA_results.pdf (137.9 KB, 62 views)
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Old 02-02-2015, 11:15 PM   #2
nucacidhunter
Jafar Jabbari
 
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It depends on the kit that you are going to use and amount of DNA available. You can do size selection first and use at least half the recommended input amount for library prep. Library can be prepared with lower amount as well but that would affect library diversity and coverage uniformity. There are some kits for low input DNA below 10 ng.
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Old 02-19-2015, 10:10 AM   #3
Hamid
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Please check the integrity of your DNA samples by running some unsheard samples on an agarose gel. Also please note that bioanalyzer chips are very sensitive to contaminants. How were these DNA samples purified?

Thank you

Hamid
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