A very easy method is just to take a clean pipet tip and gently poke the band that you want to reamplify.  Then just touch that tip into the reaction mix in a tube where you have set up the secondary PCR reaction.  Avoid transferring any visible amount of agarose.  Also, use a long-wave (low energy) UV light source to visualize the band in order to minimize any UV-induced damage.  Because the concentration of template is so high in that gel band, you need to only transfer extremely small "invisible" quantities of it to provide ample template to your secondary reaction and there is no need to purify a large quantity of it unless you want it for some other purpose.  (Also see my 2nd post below for a variation of this, which allows you to save something for storage.)

You can use any PCR purification kit as for e.g. https://www.qiagen.com/gb/shop/sample-technologies/dna/dna-preparation/qiaquick-pcr-purification-kit#productdetails

Hi Sikder, 

Run the PCR product on a gel and cut the band using a gel extraction tool (example: http://www.sigmaaldrich.com/labware/products/x-tracta-gel-extraction.html). You may want to lower the amperage and increase the run time of the electrophoresis to make sure that any primers that are left in the PCR primer are not included, or in the region of, the band that you're trying to extract. You can then run the gel through a gel extraction kit like Manasi suggested. I've had great success with the Qiagen product they suggested. 

A very easy method is just to take a clean pipet tip and gently poke the band that you want to reamplify.  Then just touch that tip into the reaction mix in a tube where you have set up the secondary PCR reaction.  Avoid transferring any visible amount of agarose.  Also, use a long-wave (low energy) UV light source to visualize the band in order to minimize any UV-induced damage.  Because the concentration of template is so high in that gel band, you need to only transfer extremely small "invisible" quantities of it to provide ample template to your secondary reaction and there is no need to purify a large quantity of it unless you want it for some other purpose.  (Also see my 2nd post below for a variation of this, which allows you to save something for storage.)

Thanks everyone for all of these suggestion. I myself also know about that QIAquick PCR Purification Kit but i am thinking for an easy solution if available. :-) 

Okay, that's a good idea to cut the portion of gel that contain the pcr product but can silica column used in DNA extraction can help to isolate that pcr product from that agarose gel suspension ?

One more important thing, during  loading in to gel, i used loading dye and gel was staining by Etbr. So, Do these component can make any trouble in using that pcr product as template in second pcr ? I am saying as because we all know that Etbr bind with DNA.  

Hi Sikder, 

The gel extraction kit made by Qiagen (https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/qiaquick-gel-extraction-kit#orderinginformation) will remove the loading dye and gel stain (it's made specifically for that). 

For my own research I wanted to concentrate eluted DNA and contacted Qiagen about using the columns for that purpose (I primarily use their DNEasy Blood N Tissue kits). They strongly recommended against using the same column because I wouldn't be able to adjust the pH properly. The kit I linked above actually accounts for any adjustments in pH needed and should work well for your purposes. 

Cheers,

Jake

Another variation that I have used successfully is to use a plastic pipet tip to stab and collect a small piece of agarose containing a small piece of the band.  Transfer to 50 ul of water in a PCR tube.  Boil the sample for four minutes in a thermal cycler.  Then use 2 ul as template for a subsequent PCR reaction. 

The attached picture shows a gel with bands that have been "picked".

Dear brother, you can keep your concentration on purification kit and please take a help of Google scholar .I hope you will get so much ways .

Thank everyone so much. Hope i can apply all these method to get a good result. :-). Thank you all.

-nahid Islam

Elution of PCR product from gel is the best way, but in this you have to cut the desired band and when you have multiple or non-specific bands in your gel. As your gel has only one band around 470bp, you need not require to run the gel. Instead, you can directly purify the gene of interest from reaction mixture itself by using same gel purification kit and it will remove the primer dimer and other impurities.

I always use this procedure for reaction mixture cleanup after restriction digestion when the size of second band is very small.

It will help to you.

Hi David, 

I had a question about your method. Does the agarose interact/inhibit subsequent PCR reactions? This could really streamline my nested PCR pipeline.

Thanks, 

Jake

I agree with Manoj. 

When I run a PCR, I will only perform gel extraction if there is a large amount of nonspecific product. Since you have only one band, you can simply perform a simple PCR cleanup using a kit. It will only take 15 minutes.

If you do not have the kit, you can perform a Phenol-Chloroform extraction (to get rid of your polymerase and other proteins) and then an ethanol precipitation. Primers are short and will not precipitate as well as your PCR product.

@Jacob Price  If you follow the plan in my second post above--poke, boil in water and use a small portion as template, you should not see inhibition of your PCR.  Usually, when I've done nested PCR, with both primers different in the second reaction, it has only been necessary to dilute the primary reaction by a very large factor like 1/1000 or even 1/10,000.  I only used the poke/boil method when the product of the primary reaction had multiple bands, as shown on the photo attached to my previous post.

Dear David F Barker,

Okay, if i poke the band and boil it, then Is this boiling will make that DNA intact ? .As we know in 95C temperature DNA denatured (like why we know in view of pcr amplification protocol) so, when the poked (Gel+ Desire DNA) band boil in roughly 100C, will it make any change in trample DNA or denatured it ?

@Sikder Nahidul Islam Rabbi:

Yes, the DNA will denature when you boil the agarose bit and the boiling will melt the agarose and release it.  (By "boiling", I mean heat at 95C.)  Remember that the first step of a PCR is also "boiling" to denature the DNA and make it single-stranded.  So heating the agarose pick will not affect the subsequent PCR reaction--its just that the DNA that you add to the reaction will already be single-stranded.

Dear David f Barker Sir, 

Thanks for your illustration and as being an easy method so i should try it. Hope to get a good result.

Thanks everyone for all information you have shared to make this problems solve. I hope, following your process i can find a good solution. Thank to Researchgate also, for making a platform, where young researcher or student like can learn and reach to Scientist and Research of renowned Institution.

Dear brother,

I am also agree with David f Barker Sir .I have read all of his posts.His words are really good for understanding the process .I think you can take a partially help of youtube  for seeing the videos .I  searched for yours and saw some videos .So i think it will be helpful for you .

Regards,

Ashrafudoulla

dear brother ,if you want i can contact with Professor Dr. Mostafizur Rahman former professor of Dhaka University .Now he is working in Manarat International University as Director of bio medical research center .I think he can help you  a lot on this topic .

Sorry I was travelling and just saw your question now. Since you have only one band and have secondary PCR primers, in my opinion, you do not need to run an agarose gel. You can simply digest the PCR product with ExoSap-IT to get rid of the unused Pair 1 primers and do the second PCR with new primers.

Dear Yenhui Chang,

Madam, Thank you so much. :-)

Even i didn't know anything about ExoSap-IT, as you have suggested so i Google it got information about the product (ExoSap-IT) for pcr easy clean up without any degradation or modification of that existing pcr product in the tube.Excellent. 

Thank you so much, hope it will help me a lot.

You are very welcome! ExoSap-IT is very easy to use. It takes 15 minute to digest the primers and 30 minute to inactivate the enzyme. Make sure adding enough ExoSap-IT or dilute PCR product so the digestion is complete.

Please find attached protocol. My lab uses ExoSap-IT all the time. It takes 15 minutes at 37C to digest the PCR product and 15 minutes at 80C to inactivate the enzyme.  It can be ordered from USB.

Dear Yenhui Chang,

Thank you Madam for all of your information and attached brochure. :-), i have read it and yes its an easy method and need less time for purification . :-)