it is not a answer to your question but if you designed nested primers  in the amplicon of interest and specific to that amplicon you could just run another round of pcr to get much and clean amplimer

Thank you very much for your Answer Paul, I had thought about Nested PCR but the issure is that i do not know the sequence of my amplicon (I should have specified it). I am using very degenerate primers to amplify a viral gene and i don't exactly know what my bands are (I observed from previous sequencing that some bands are just genomic DNA resembling viral sequences which get amplified but i cannot discriminate which are viral or not before sequencing).

I could try to designed a set of Nested primers for the viral gene, but i am not sure to be able to do that, it was already very difficult to design the primers i used.

Hi, in our lab we are using Gene-JET gel extraction kit. https://www.thermofisher.com/order/catalog/product/K0691. Works great! Best wishes for you.

Hello Umberto,

We are using the Agarose gel DNA extraction kit from Roche. This works quite well for us.

Hi,

We work with QIAquick Gel Extraction Kit; has worked perfectly fine for us over the years.

Thank you everybody for your answers. Jörg, may I ask you if you have modified the protocol? We are loading few pcr product (20 ul) and following the protocol and we keep obtaining few, unpure DNA. If we load more pcr product (> 60 ul) then the kit works fine.

Thanks

Hi Umberto,

I am afraid there's the rub!  We use the QIAquick kit to sequence gel separated PCR products. We load as much as we can onto the agarose gel and follow the protocol as is. We did not have any problems. We do not have experience with this kit, when the band on the gel is e.g. dim, only.

In  your case I would call Roche to have sent you a sample (for free) of their extraction kit and try it out.

Good luck, Jörg

Hello Umberto,

Qiagen, Michel Nagel,  Roche, and etc. kits are good. I think you should not spend the time to try different kits. You can try to increase your templates by running more PCR reactions, loading more gels, and combining all of them in one column of gel extraction kit. I believe you will get good results.

Good luck,

Thuc Bui

Thank you all for your valuable answers, we will try Roche Kit and see if we encounter the same issues, in that case we'll resign ourselves to load more PCR product on the gel.

Thank you, Umberto

While trying the recommended kits, I suggest RNAzol extraction. It is labor intensive, but since you know how much DNA you need, careful processing of your sample will yield more than enough for your subsequent processes. Good luck.

I used to use 1% low melting agarose and used gel extraction kit from Qiagen.Worked well for me!

you could also dispense with the kit .Just run the sample in low gelling agarose ,cut out the band you want, freeze it a couple of times to get the liquid  from the agarose and re amplify it. Hard to get the number of cycles right but if you amplify  some tubes between about 8 and 16 cycles  you should get clean amplification and plenty of product in a couple of them

Hi, 

You can use this kit from Qiagen

https://www.qiagen.com/us/shop/sample-technologies/dna/dna-clean-up/qiaquick-gel-extraction-kit/#orderinginformation. 

Hi Umberto,

our kit of choice is the illustra GFX PCR DNA and Gel Band Purification Kit from GE. But I would like to recommend you another easy and often successful method. Just pick the fragment of interest on the gel with a fine 10µl pipette tip for one or two times and cut about 5mm (more doesn't matter) into a new reaction tube. This is now your template for a second round of PCR. Sufficient amounts of Template DNA will be released during the first denaturation step while the low amount of agarose doesn't inhibit the reaction in most cases.

Good luck Dietmar

http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11770&catalogId=10101&productId=19608&storeId=11762&langId=-1

I thank you all for your answers! Paul and Dietmar, we will for sure try the methods you have suggested, they are both very good ideas which i still haven't tried.

good luck. Excision works best on low gelling agarose as ordinary agarose inhibits pcr  or if using ordinary agarose use a high dilution of the product and a few more cycles for the same reason

Hi Umberto, Use gel prepared by low melting agarose for running the PCR products. Use QIAquick gel extraction kit by Qiagen, it works well. Just use half of the elution buffer in coulmns in the final step of eluting the DNA and wait 3 minutes after adding the elution buffer. Then proceed with spinning the column. You should get good yield of DNA. 

I use Gel purification kit by Thermo Fisher Scientific...It's easy and quicker...It has Binding buffer, Washing buffer and Elution buffer for gel purification...Just 15-20 minutes of protocol...

I used Gel purification kit by Bio & Sell, also tried GeneALL Kit. But the best results observed with the 1st one. It is very efficient, quick to use protocol and better results

Dear Umberto,

We used the following kit: Wizard® SV Gel and PCR Clean-Up System Protocol - Promega. It is an efficient and with an acceptable cost, moreover we use this kit to purify selected amplicons for further sequencing.

Regards

Dear palatini 

Happy to inform you what would be useful for  DNA extraction from Agarose Gel. i our lab we highly use cosmogenetech product and its gives high quality with high concentration of DNA

http://www.cosmogenetech.com/cosmo/productmngr/prm17.jsp

Hi, we use with good yield rhw Wizard® SV Gel and PCR Clean-Up System (Promega) to clean PCR bands from DGGE.

QIAquick Gel Extraction Kit works well for me

As was mentioned, Promega and Qiagen have the best..

Hi Palatini 

Thermo scientific GeneJET Gel Extraction Kit is very fast and convenient.