Hey Sergios,

whenevever it comes to PCR product or gel extraction cleanup. I use NucleoSpin Gel and PCR Cleanup from Macherey & Nagel (http://www.mn-net.com/Products/DNAandRNApurification/Cleanup/NucleoSpinGelandPCRCleanup/tabid/1452/language/en-US/Default.aspx)

We use this when cleaning up extracted agarose gel bands in preparation for NGS (Illumina sequencing).

To maximize DNA yields we tweaked the clean-up protocol a little by adding an additional elution step (elute the DNA two times with the same amount of elution buffer/TE/H2O or the like).

We have had good experience with this Kit so far and I can say that it has worked for our NGS purposes.

Good luck!

I just used Bioline's Isolate II (http://www.bioline.com/us/isolate-ii-pcr-and-gel-kit.html) and the gel bands fainted a lot yielding low quantification readouts, i.e. we lost a lot of PCR product.

I used to use Agencourt's AMPure XP magnetic beads (https://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/nucleic-acid-sample-preparation/agencourt-ampure-xp-pcr-purification/index.htm#2/10//0/25/1/0/asc/2/A63880///0/1//0/), but I am examining cheaper solutions.

Hey Sergios,

whenevever it comes to PCR product or gel extraction cleanup. I use NucleoSpin Gel and PCR Cleanup from Macherey & Nagel (http://www.mn-net.com/Products/DNAandRNApurification/Cleanup/NucleoSpinGelandPCRCleanup/tabid/1452/language/en-US/Default.aspx)

We use this when cleaning up extracted agarose gel bands in preparation for NGS (Illumina sequencing).

To maximize DNA yields we tweaked the clean-up protocol a little by adding an additional elution step (elute the DNA two times with the same amount of elution buffer/TE/H2O or the like).

We have had good experience with this Kit so far and I can say that it has worked for our NGS purposes.

Good luck!

Invetrogen and favorgen kit

Like Sergio we use AMPure XP and were recommend to do so by Illumina.

I just procured DNA cleanup kit from agarose and DNA cleanup kit from PCR samples from Jena Biosciences Germany. It is very cheap and the yield is very good, also the procedure takes less time.

Like sergio and Michelle, AMPure from beckman for me is best. Specially, when you want to avoid huge number of PCR duplicates after in NGS, then you make low number of PCR cycles and having much less PCR products, purification in solution (even you can do size selection) by AMPure is really good. Anyway, thé yield of AMPure (DNA recovered) is largely higher than any column.

I would also recommend the magnetic beads-based Agencourt AMPure XP - PCR purification from Beckman Coulter

https://www.beckmancoulter.com/wsrportal/wsrportal.portal?_nfpb=true&_windowLabel=UCM_RENDERER&_urlType=render&wlpUCM_RENDERER_path=%2Fwsr%2Fresearch-and-discovery%2Fproducts-and-services%2Fnucleic-acid-sample-preparation%2Fagencourt-ampure-xp-pcr-purification%2Findex.htm#2/10//0/25/1/0/asc/2/A63880///0/1//0/

I agree with Patrick that NucleoSpin from Macherey & Nagel are the best. However, most commercial kits should be good enough for NGS. I have personally used GeneJet from Fermentas.

Jesper

What about an enzymatic cleanup, like ExoSAP-IT (Affymetrix)?

http://www.affymetrix.com/catalog/131310/USB/ExoSAP-IT-For-PCR-Product-Cleanup#1_1

I would recommend this one

http://www.mobio.com/images/custom/file/12500(1).pdf

Good luck,

Qiagen

Hello Sergios,

I have also worked with ExoSAP-IT. I think it can be used to rapidly clean up PCR products, when you just aim to quickly sequence a sample (lets say after a 16S PCR). I wouldnt recommend to use it for NGS based approaches, since the enzymatic cleanup only reduces remaining PCR additives, but may fail to completely digest e.g. primer dimers.

If you want to perform NGS based on a specific region of the 16S rRNA gene (V3 , V6 or whichever region u amplify), I recommend performing your PCR and then load all of your PCR product on an analytic gel (use high quality agarose, so that you get distinct bands for the PCR products and avoid contamination by e.g. primer dimers) and then perform gel extraction using a clean up Kit (after you cut out the band from the gel).

This way you can ensure, that only the region of interest and no contaminating side products are sequenced in your NGS approach.

In essence, I do NOT recommend using ExoSAP-IT or similar products as your only clean up procedure before NGS!

I vote for the Macherey Nagel Nucleospin kit - works great for us!

Personally I use the Gel Extraction kit from Qiagen. Works fine and I get good DNA elution (Qubit measurement). But I have to say that my bands are very good visible in gel, means a lot of material. Downstream NGS analysis worked fine.

I worked both Marchery Nagel and Qiagen cleanup kits as wel as XPbeads. A: I used it for PCR cleanup : i see better cleanup with the MN kit vs qiaquick but XP beads is best but works a bit troublematic when starting with bigger volumes. So if i pool many samples before cleanup i first concentrate with the MN kit and subsequently cleanup by XP beads (for NGS purposes). B: I use XP beads to concentrate gDNA, Qiaquick also works reasonably but somehow the MN PCR cleanup kit cannot capture bigger sized DNA very well.

My advice; https://worldwide.promega.com/-/media/files/resources/protocols/technical-bulletins/101/wizard-sv-gel-and-pcr-clean-up-system-protocol.pdf?la=en

HI Sergios-Orestis Kolokotronis

Zymo's DNA Clean & Concentrator is the best PCR clean up kit!https://www.zymoresearch.com/collections/dna-clean-concentrator-kits-dcc

-Elute in as little as 6ul to get it super concentrated

-Minimal losses (>90% recovery)

-About 20% Cheaper compared to other kits in the market

-FREE samples: https://www.zymoresearch.com/collections/dna-clean-concentrator-kits-dcc/products/dna-clean-concentrator-5#

Cheers,

Luigi