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-   -   DNA extraction from Saliva (http://seqanswers.com/forums/showthread.php?t=30050)

Anna IT 05-08-2013 09:50 AM

DNA extraction from Saliva
 
Hi everybody!!
I have to extract DNA from Saliva, in order to realize some ION Torrent PGM libraries. I'd like to ask if you know which is the best kit for DNA extraction. In the past we used the Oragene extraction protocol, but our samples were full of proteins. So we have thought about using the DNA mini blood kit, that is compatible also with saliva. In fact our aim is to have a good concentration, but also a great quality of DNA samples. One of the limits of this kit is the cost, because it's really expensive. So I tried to find out something similar, but less expensive. I've read something about the NORGEN DNA saliva isolation kit. Have you ever used it? Do you know other kits that I can use?
Thank you in advance for answering!
Anna

jwag 05-08-2013 06:56 PM

My lab always uses the Qiagen DNeasy blood & tissue kit for extracting DNA from tissues. We used it for prepping DNA for Illumina sequencing and it worked well.

james hadfield 05-09-2013 01:59 AM

We've made NGS libraries from Oragene DNA and 23andMe used it for my genome. Oragene is expensive but you get masses of high-quality DNA.

here's a list of providers I am aware of:
Oragene. http://www.dnagenotek.com/US/product...ctionkits.html
Norgen. http://norgenbiotek.com/display-product.php?ID=493
nexttec 1-Step. http://www.nexttec.biz/products
PSP SalivaGene http://www.invitek.de/products_and_s...agene_dna_kit/

I'd be intersted to hear if anyone has compared saliva extractions kits.

MikeTayeb 05-16-2013 07:38 AM

Hi Anna

We've used Oragene for NGS on both the Ion Torrent and Illumina systems (GAII and HiSeq2000), both targeted and whole genome and we didn't have any problems. We always extract using prepIT-L2P (formerly Oragene Purifier), an EtOH precipitation-based method, and we don't have problems with protein contamination.

Are you assessing protein contamination by 260/280 ratio? If so, you should be able to obtain 1.7 or higher in most cases with a prepIT-L2P extraction. Column-based extraction like prepIT-C2D or various Qiagen kits might give you a bit higher ratio (1.75 or 1.8) but you'll sacrifice yield and, in my experience, it's not necessary to use column extractions for NGS.

Mike Tayeb
Manager, Technical Support
DNA Genotek (manufacturers of Oragene)

ECO 05-16-2013 07:46 AM

We've substituted saliva into Qiagen whole blood preps with good results, but be aware of how much bacterial DNA will be in saliva preps.

Chris Slowey 12-12-2013 09:01 AM

Saliva Devices
 
Hi guys,

Often times the collection can be the key to getting a pure and high quality sample. I work for a company that produces a range of saliva collection devices, I think you might find the DNA·SAL™ : http://4saliva.com/products/dna-sal/ a perfect fit for gathering a good, clean, and simple sample.

We are a small company really focused on customer service, we are really happy to help anyone through any issues or questions they might have. We go to great lengths to customize our products where there is need as well. Hope this helps.

ctseto 12-18-2013 04:47 AM

There's a NEB kit that removes human DNA for microbiome workflows; NEB has a protocol to elute the human DNA.

https://www.neb.com/products/e2612-n...enrichment-kit

https://www.neb.com/protocols/2013/0...host-dna-e2612


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