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 DNA Purification: Phenol Chloroform X PEG 30% [View Printable]
Leprevost

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Hi

I have a doubt about DNA purifications.
Im working with Gateway system, and now I have to purify my PCR products. The Gateway protocols tells me to use a fast purification protocol using PEG, basically for removing dNTPS and oligos <300 bp.

So, I have two questions abou it.

Can I remove dNTPS and oligos from my samples with the basic phenol extraction protocol?

and

It is possible that a phenol residue in my sample interfere with my attb / attp recombination?

Thanks
.........................
IBMP - Paraná Institute of Molecular Biology
http://www.ibmp.org.br/

 Posted Mar 17, 2007, 5:56 AM
vanishing

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the best thing to do is use colums to purify that

lots of products out there

e.g.:

http://www.clontech.com/products/detail.asp?tabno=2&product_id=10560

http://www.stratagene.com/products/displayProduct.aspx?pid=394

http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx

since you are using the gateway system, money cannot be an issue, can it?
.........................

Posted Mar 19, 2007, 3:46 AM
omehenk

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I agree with vanishing: stay away from phenol extraction. I've used gateway a lot and standard pcr spin columns are usually sufficient. If your gels show contaminating PCR bands it is probably better to gel purify (again, use a qiagen kit or similar).
The annoying thing about gateway is the huge size of the primer tags, which makes spin columns less efficient in removing primer oligos and/or primer dimers. However, As long as these are not visible on a gel after purification you should be alright.

We've now switched to in-fusion cloning though, much better and cheaper.
.........................

Posted May 02, 2007, 12:28 PM
Tony Rook

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