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-   -   RNase treatment (http://seqanswers.com/forums/showthread.php?t=7626)

wormseq 11-01-2010 12:49 PM

RNase treatment
 
Hi all,
I have several genomic DNA samples that were extracted using a Qiagen DNeasy kit without the optional RNase treatment that I would like to submit for sequencing (75 bp paired-end libraries for Illumina). I have quantified the samples using a PicoGreen assay and there appears to be plenty of DNA for sequencing. However, I was wondering if there is any reason to be concerned about the residual RNA in the sample interfering somehow with library construction? Any thoughts on this would be greatly appreciated. Thank you.

chma1112860 09-05-2013 08:11 AM

Hi wormseq

I currently wonder the same thing.
Do you find any answer ?

Martin

kwaraska 09-05-2013 09:37 AM

I wouldn't worry too much. The protocols are pretty much designed only to work with double stranded material. I might try and figure out the percentage of the RNA and adjust your DNA concentration accordingly so you put the correct amount of starting material into your prep.

chadn737 09-05-2013 09:43 AM

If you are worried about it, why not throw in a little RNase, let it work its magic for a while, and clean up your samples? You shouldn't loose much, particularly if you use a Phenol/Chlorophorm extraction to clean up your samples.

chma1112860 09-06-2013 12:13 AM

Thanks for your answers.

Actually we have about 600 gDNAs already extracted without RNase treatment.
I just wanted to know if it is absolutely necessary to eliminate RNA before starting the library preparation, to avoid useless long work.
We plan to use a Nextera XT kit after making several amplicons of differents sizes.

I think that if we position primers in intronic regions, we won't have amplification artifacts owing to the presence of RNA.


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