Harindra, as some of the others have already stated, using the standard PCR or gel extraction kits will both clean a PCR product sufficiently for downstream applications, but which to use depends on what you wish to do. If you are absolutely sure you produce a single product from your PCR, you can skip running the whole sample on a gel and only run a small fraction to validate the PCR reaction. The rest of the sample can be purified using the PCR purification kit. On the other hand, if you are not sure or additional minor products are produced from your PCR reaction, it is best to separate the PCR products from the reaction using an agarose gel, excising your band of interest, and then using the gel extraction kit to purify your DNA. In the second case the only reason to use the PCR purification kit is, as others have stated, to concentrate a sample so that all the reaction product can be loaded into a single well of the gel. An additional word of advice, most kits come with an elution buffer which is Tris pH 8.0. This or distilled/MQ water is fine if you wish to carry-out downstream procedures such as sequencing. Some older protocols call for TE. It is best not to use TE as the EDTA can interfere in some downstream procedures.

No you do not have to for a gel run, you only need to do this if you are taking your PCR product through a downstream application such as cloning or sequencing.

Thanks Bassam! So I can just load whatever I'll get from the PCR reaction to the gel, then extract the gel using a gel extraction kit or so which can be sent for sequencing right?

No it's not necessary. In most cases the gel extraction kits contain a salt-ethanol wash step to clean the DNA, which is pretty much what the PCR purification kits do.

Thank you both, appreciate your help. I was always wondering why there are PCR purification kits as well as gel extraction kits. So they both do pretty much the same right?

@William, Since I am planning to use a gel extraction kit ( after the gel electrophoresis) to purify or extract the band I am interested in, so there is no need to use an additional PCR purification kit to purify DNA from the PCR mix before loading to the gel? Would the gel extraction kit do enough for the subsequent sequencing?

One of the advantages of using PCR purification before gel extraction is drastic reduction in volume of your sample (and, therefore increase in concentration). Extras includes ssDNA (oligos), salt and protein removal. if your initial PCR prep is large (200-300 ul) it is good idea to do PCR purification Kit first, before applying the DNa nto the gel for gel-extraction procedure. Of course, you can always do Phenol extraction followed by EtOH precipitation instaed of column PCR purification, but that will take more time.

The gel resolution is way better if you apply DNA sample that does not have salts and/or proteins.

It seems a bit unnecessary to go through loading your whole product onto a gel to visualise it, then gel extracting the whole thing to purify (unless of course you're expecting multiple bands, and wish to just excise the one of interest - in which case ignore the rest of my answer).

Normally you should just run a small aliquot of your pcr product on a gel to visualise it, and then if all looks good by gel you can purify the rest of your pcr product ready for sequencing. No messing around with gel extractions.

As far as I believe and my experience says One should always perform purification if you are looking for Sequencing. This helps in getting high quality data.

Thanks Dhrumit, So you say, I should purify PCR products, then load the purified products in to the agarose, run the gel, cut the band I'm interested (Hopefully there will be just one) and use a gel extraction kit to further purify and send them for sequencing?

It is recommened to run a gel post PCR for mainly 3 reasons:

1- to check if ypu have sucessful amplification and you have enough of it

2- to check if you have amplified the right fragment size

3- if you have primer-dimer issues and it is dificult to remove by PCR clean-up kit such as EXo-SAP

Depending on your PCR volume, i would not recommend using more than 10% of your total PCR volume. If all is good, i.e. No primer-dimer and right fragment, then use the reminder volume in a standrd PCR clean up kit (filtration or mag beads based chemistries)

Harindra, as some of the others have already stated, using the standard PCR or gel extraction kits will both clean a PCR product sufficiently for downstream applications, but which to use depends on what you wish to do. If you are absolutely sure you produce a single product from your PCR, you can skip running the whole sample on a gel and only run a small fraction to validate the PCR reaction. The rest of the sample can be purified using the PCR purification kit. On the other hand, if you are not sure or additional minor products are produced from your PCR reaction, it is best to separate the PCR products from the reaction using an agarose gel, excising your band of interest, and then using the gel extraction kit to purify your DNA. In the second case the only reason to use the PCR purification kit is, as others have stated, to concentrate a sample so that all the reaction product can be loaded into a single well of the gel. An additional word of advice, most kits come with an elution buffer which is Tris pH 8.0. This or distilled/MQ water is fine if you wish to carry-out downstream procedures such as sequencing. Some older protocols call for TE. It is best not to use TE as the EDTA can interfere in some downstream procedures.

Dear Harindra,

a very nice and comprehensive explanation by Dr. Blalock.

You can stick to a single procedure using gel extraction kit, which will serve all the three purposes as pointed out by Dr. Elfahamwi, as you can visualize what has happened in the PCR reaction. Because, every time you keep a reaction, not necessary that your amplification efficiency will remain the same which may be due to technical or handling issues. Sometimes variation is seen even in well trained hands, so no worry. At the same time you can also purify the PCR product by cutting out the gel piece with your expected band that you actually visualize. But note that, all kits do not give the same efficiency of DNA recovery after purification, so start with multiple PCR reactions (replicates). Do not use more than 50 microlitre per PCR reaction, or follow the manufacturer's protocol of the polymerase you are using. Do not heat the column while eluting DNA with water in the last step of gel purification. And finally as Dr. Blalock said, use water instead of TE buffer. While sending for sequencing, try to have a concentration of 200 ng/microlitre for better result and send no less than 15 to 20 microlitre. Anyway, the company whom you give will tell you about the details of DNA concentration, amount (volume) and primer details (concentration and volume).

Good Luck !!!