you can simply run the PCR product on a gel then , the cut the gel containing the band you need , and perform a DNA- from - Gel extraction protocol, there are protocols available for that

you can simply run the PCR product on a gel then , the cut the gel containing the band you need , and perform a DNA- from - Gel extraction protocol, there are protocols available for that

I agree, if you're already running your product in a gel, a gel extraction of the bands is the next step.

Your university likely already has the needed kits to do this, ask around in molecular biology labs for a "Gel Extraction Kit" or suggest buying one like these.

http://www.zymoresearch.com/dna-purification/dna-clean-up/gel-dna-recovery/zymoclean-gel-dna-recovery-kit

are you doing multiplex pcr? Amplifying 3 products in one reaction?

You can go for gel extraction or in case you have funding you can clone it into PGEMT vector and sequence the plasmid which would be a better option. If you are getting multiple bands with same primer you can cut the gel (your band of interest) and immerse it in pcr grade water for about a day and then use this as a template.

Run agarose gel cut the bands in your product size range, gel extract using GFX columns or qiagen columns, TA clone into pGEMTeasy vector system. Its easy to sequence from pGEMTeasy vectors using T7 SP6 primers and once you confirm the sequence, the template is available for further cloning procedures and importantly, sequencing from plasmids will give clean sequencing peaks than a direct PCR product sequencing.

I completely agree with Dr. Goodwin. We follow the same protocol in our lab using pGEM-T easy vector and it's a very promising and easy way for PCR product sequencing . But in addition I want to tell one thing that your PCR product should have poly A tail for cloning in this vector. If you are amplifying your product with Taq polymerase then the products already consist the poly A tail. But if you are using Pfu polymerase then you have to add poly A tail later to your PCR products. You can find the complete protocol here:

http://worldwide.promega.com/~/media/Files/Resources/Protocols/Technical%20Manuals/0/pGEM-T%20and%20pGEM-T%20Easy%20Vector%20Systems%20Protocol.pdf

Best of Luck !!

If you are getting a single product without any other bands you can simply use PCR purification kits,its a simple protocol to follow and then do sanger sequencing.Secondly there are tips available, simply pass your product through those tips you will get the purified product.

But if there are more than one band use gel extraction method.

I start with your PCR product :-

First you run PCR product on 1.2% agrose gel with ladder cut desired band and weigh the gel ,and purifies with Quigen PCR purification kit.

1-Add three times QG-buffer(gel weight),and incubate at 50 degree centigrade for 10 minutes or completely dissolve gel .

2-Add equal volume (gel weight) Isopropenol and wait for 5 monutes for precipitation.

3-Add this solution in column and run at 13000rpm for 60 seconds.

4-Add 750 microliter of QG buffer for elusion of remainig gel,run at 13000 rpm for 60 seconds.

5-Add 750 micro liter of PE buffer and run at 1300rpm for 60 seconds.

6-Run blank at 13000 rpm for 2 minutes.

7-Open the mouth of column and wait 2 minutes for evaporation of ethanol .

8-Then take a fresh autoclaved 1.5 ml centrifuge tube and put the column in fresh tube and add 50 microliter of elusion buffer(EB).

9-Store at -20 degree centigrade .and measure the concentration of Purified DNA with Nanodrop.

As others have said, cutting the band out of a gel if you have multiple products is quick and easy, or if you have only a single product, a quick filtration.

If the only thing you want to do with the PCR product is to sequence it, I wouldn't bother with subcloning into a vector. The pGEM vectors suggested certainly are great tools, and if you need good sequencing data for the entire PCR product, you should use them. However, if you only need sequence in the middle of your product (which is what it sounds like to me from your question), the extra work to ligate, transform, grow E. coli and isolate the plasmid isn't worth the bother if all you want is the sequence data. Just clean up the product and use your PCR primers for the sequencing reaction. If you need large quantities of the product for other purposes then a TA vector is the way to go and gives you more options for sequencing primers.

Depending on your product and what you're trying to get, you have a couple options. Like everyone has said, you can run your product on a gel and perform a gel extraction. I'd recommend this method particularly if you have multiple bands or a lot of primer-dimer, so you can ensure a clean template for sequencing. If all you're planning to do is sequence, then cloning is probably not necessary, as Eric Patterson said above; you can go straight from the gel extraction to a sequencing reaction.

If the product is a single, clean band, I've used enzymatic cleanup with exoSAP (http://www.affymetrix.com/estore/browse/brand/usb/product.jsp?productId=131310#1_1) to prepare for sequencing and still got good data.

It is even simpler to use ExoSAP. Simply add 1 microL to 2,5 microL PCR product incubate 15 min at 37C followed by 15 min at 80C. Then you can do the seq.- reaction with a relevant primer (2 reactions per sequence if you make it in both directions).

We are doing this routinely (4000 seq/year) from a multiplex PCR with up to 4 different PCR products and it works.

I completely agree with Dr. Naveen Sathyan and Dr. Debaprasad Parai. If not handy kit to extract DNA from the gel, you can use the filter tip, a prior cut off the edge. Add to it from the gel fragment. After that, use a set of pGem-t.

After 3 ul electroforesis in 1% agarose evaluation, we purify the rest of the PCR product by precipitation with an qual volume of 20% Polyethylene glycol in 2,5M Sodium Chloride. Mix well, incubate 15 min at 37ºC ,centrifugate 15 min at 13.000 x g, discard supernatant with pippete. Gently wash the colourless pellet with 70% ethanol, centrifuge 3 min, discard ethanol and let the pellet dry 15 min at 37ºC. Redisolve in sterile double distilled water.

Dear, you run 1% agarose and run at low voltage for 1 hour, this make band difference then excise required bands purify it by gel purification kit (thermo) if after purification good band then send for sequencing. be happy

PCR purification kit from qiagen is very effective. you don't have to run it on a gel, just apply PCR product to filter and follow protocol.

however, if your lab doesn't have a kit, and you can't use someone else's kit...then run on low-melting point agarose gel [NOT standard agarose], the voltage applied will need to be lower, too. then, excise the band and melt in 65C water bath. add 5 volumes phenol...reheat as addition of phenol will cause the agarose to re-harden. then follow standard phenol:chloroform DNA extraction, or whatever DNA extraction methods your lab uses.

better you go for gel extraction using Qiagen gel extraction kit.

If you shouldn't have access to a gel extraction kit, you can also order test kits from most companies sufficient for 5-10 extractions with no charge, so if it's a one time thing and you have just a few products you want to purify, this could be a cheap way.

For sequencing, I have used the kits from Macherey Nagel and from Roche (High Pure PCR Product Purification Kit) with very good results. In both cases, you don't need to excise the band from the gel, just purify the PCR product. If you have several bands I recommend you to run the first product on a gel to visualize the bands and with a clean needle "stab" the band of interest and run a second PCR to reamplify.

I agree with Anja Kovanda.

The E-Gel® CloneWell works nice. Try to purify the pcr products using it.

You can simply cut with amplicon band from gel and transfer into a homemade column. The column can be cut in half from the spout for a pipette with filter and tube type Eppendorf