DNA Purification: Phenol Chloroform X PEG 30%

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Leprevost
Leprevost's picture
DNA Purification: Phenol Chloroform X PEG 30%

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

vanishing
vanishing's picture
the best thing to do is use

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?

omehenk
omehenk's picture
I agree with vanishing: stay

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.

Tony Rook
Tony Rook's picture
For those of you interested

For those of you interested here a some links to Clontech's product pages for

In-Fusion PCR Cloning Kits

"In-Fusion PCR Cloning Kits allow you to clone any PCR fragment into any vector in a single step without restriction digestion of the PCR fragment, ligase, or blunt-end polishing. The method relies on our proprietary In-Fusion Enzyme, a protein that can fuse PCR-generated sequences to linearized vectors efficiently and precisely. All thats required is a simple 30 minute incubation."

OR

In-Fusion 2.0 PCR Cloning Kits

"The In-Fusion 2.0 PCR cloning system allows you to clone into your standard vectors, at any restriction site, with ease and high efficiency. Precise clones can be generated without PCR product purification, restriction digestion, or blunt-end polishing, and the cloning is ligation-independent. The In-Fusion 2.0 method relies on direct treatment of PCR products with our proprietary Cloning Enhancer, and the In-Fusion Enzyme, a protein that can fuse linear DNA with small regions of overlapping sequence efficiently and precisely. All this is achieved in a simple short reaction that generates the exact clones desired in just two days.

In-Fusion 2.0 cloning is typically more than 5-fold more efficient than the original In-Fusion Cloning process, and is validated for fragment lengths less than 0.5 kb up to 12 kb. This highly efficient cloning process minimizes the screening required to identify correct clones. This methodology can work very effectively for single reactions or high-throughput applications.

The In-Fusion 2.0 kits come with individual tubes of lyophilized dry-down reagents to assist in quick and easy cloning. These convenient tubes can be stored right on the lab shelf. Performing a cloning reaction is very simple. You just generate a PCR insert using a DNA polymerase such as Advantage HD, then treat a portion of the PCR product with the Cloning Enhancer. After treatment, transfer the PCR insert and vector to the Dry-Down reaction tube in a total of 10 μl, and incubate. Then transform, and screen the resulting colonies for inserts.

The final product is the desired clone, with the insert in the correct orientation and no additional bases added. The ability to clone directly into any vector, including any vectors in your freezer, at any restriction site, eliminates the need for further subcloning or manipulation, and results in the exact clone required."

Please note that description of these products above are referenced directly from Clontech's product pages.