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DNA Purification: Phenol Chloroform X PEG 30% [View Printable]
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Leprevost
Group: Member Posts: 5 Joined: Jan 30, 2006
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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
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......................... IBMP - Paraná Institute of Molecular Biology http://www.ibmp.org.br/
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Posted Mar 17, 2007, 5:56 AM |
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vanishing
Group: Member Posts: 132 Joined: Apr 25, 2005
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| Posted Mar 19, 2007, 3:46 AM |
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omehenk
Group: Member Posts: 2 Joined: May 02, 2007
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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.
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| Posted May 02, 2007, 12:28 PM |
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