OK, I see. But nevertheless, you might still have DNA that is quite adequate. Bear in mind that colony PCR works with absolutely no DNA purification! If I had to purify, my approach would be to use the Qiagen miniprep kits. With phenol/chloroform, the losses will be large and you will remove just proteins and phenol (dangerous stuff!) residues will interfere with PCR. Ethanol precipitation will also precipitate protein and RNA contaminants, but it will remove salts and lipids. But given that you are probably going to amplify about 1 nanogram (or less?) of DNA, all these contaminants might not be a worry. Can you simply check that you have a decent PCR product(s) using agarose gels before you sequence? 

Did you try this?

GeneJET Genomic DNA Purification Kit

I used it for fungal DNA isolation and satisfied.

Dear Manolo

I have been research higher education for 3 decades. Sorry, but I don't know.

Beste Regardings.

The best method we have used here for different DNAs in the collection extracted with different methods is filtration in Sepharose CL-6B. Here is the protocolo attached. 

However, as you mentioned in your post, in a recent attempt of extractions for doing NGS (anchored enrichment), the first extractions did not perform well in the 260/280 and 239/260.... we just precipitated with the standard Ethanol + Sodium Acetate 3M (ph 4.6), washed twice in Ethanol 70% and ressupended.... that was sufficient to achieve purity for the protocolo. 

Thanks for your answers,

I will try Cassio's suggestion this week, and see how my samples will react.

Thanks a lot.

The Sepharose is unbeatable but the setup is not cheap.... the purification per sample results quite cheap, but the minimum you need to buy for a decent price is 1 liter, and 500g of glass beads... that is enought for 3000 purifications

What you plan to do with the DNA?

I plan to use the DNA to perform massive parallel amplification with Fluidigm's Access Array. Then I will sequence my amplicons in Illumina MiSeq.

According to the link you gave, you haven't actually extracted the DNA, but only lysed the samples. If so, the nanodrop readings are not informative. I advise to use picogreen or some other DNA-specific method if you really need to quantify the amount of DNA. The kit claims to generate PCR-ready material: why not try it as it is?

I quantified on Qubit, and I actually have DNA, but in low concentrations. I just wanted to raise the quality of my DNA because these PCRs are very expensive, and I would like to use the best DNA that I can get.

Thanks a lot for your help.

OK, I see. But nevertheless, you might still have DNA that is quite adequate. Bear in mind that colony PCR works with absolutely no DNA purification! If I had to purify, my approach would be to use the Qiagen miniprep kits. With phenol/chloroform, the losses will be large and you will remove just proteins and phenol (dangerous stuff!) residues will interfere with PCR. Ethanol precipitation will also precipitate protein and RNA contaminants, but it will remove salts and lipids. But given that you are probably going to amplify about 1 nanogram (or less?) of DNA, all these contaminants might not be a worry. Can you simply check that you have a decent PCR product(s) using agarose gels before you sequence? 

Dear Dr. Margison,

I will talk to the facility where I will perform the PCRs and the sequencing, but I think that I will be able to run my samples in agarose before sequencing, that's a great idea. Do you think that I should do this test with the Fluidigm's equipment, or a regular PCR would be enough to let me know if my samples will work?

Thank you very much for your help.

I would do regular PCRs using different amounts (say, ten fold serial dilutions) of a selection of lysates.

That's great, I will try this and see how it goes.

Thanks a lot again.

Hello all,

Just an update on my results. I followed Prof. van den Berg's suggestion to precipitate my samples with standard Ethanol + Sodium Acetate 3M (ph 4.6), and it turns out that my experiment worked really well. Most of my samples were amplified with the Access Array protocol, and the sequencing with MiSeq gave me excellent results.

The only problem that I faced in this protocol was that some of my samples were too viscous after the extraction and the purification, and these samples were problematic to load in the microfluidic reactor of Access Array. To solve this problem I just diluted 1 uL of my samples with 4 uL of water.

Thanks everyone for your suggestions.