It is not essential, but it can help. I would recommend running your first PCR product on a gel. This will give you an idea if the PCR actually worked, as well as once you gel extract the product it removes the majority of contaminants. You can spec your product, and add it to the next PCR step just as you would any normal DNA template.

I have performed nested PCR by directly taking a fraction of the first PCR reaction, and adding to the second with some success. The problem is if it does NOT work, you have several variables you need to figure out to troubleshoot the problem. If you visualize the first product and clean it up, this can minimize this optimization later.

Good luck,

~Adam

Hi Adam. Thanks for the answer. Did you clean up your product using Exo-Sap or some other technique?

"No need" is a little absolute for science in my opinion. As I stated earlier, you can absolutely try it without clean up and it may work, but if it does not then you need to troubleshoot. And to answer your question John, all I performed was a gel extraction using any standard gel extraction kit (we use Qiagen). This not only cleans up the PCR product, but when you excise it from the gel you enhance the specificity of your template by removing any non-specific products the first PCR may have produced.

Whichever way you go, just make sure you quality control at the end for the correct product. Good luck.

~Adam

From my experience there is no need to purify first PCR product for nested PCR or reamplification. But I agree with Adam, In science nothing is absolutly true or false..., you never know. So, try without purification, if it doesn't work then purify first.

Monica

For an established nested PCR protocol we always use a 1:1000 dilution of the first PCR, but during the establishment of the protocol we always check the product by gel or melting curve. Fortunately I never saw a reason to purify the product prior the nested PCR. If you are working with 96 well MTP like we do this would be a very expensive way, better to optimize the protocol of the first PCR.

I performed nested PCR for HIV diagnosis without purify PCR product and there are no problems

Thank you for all the answers. I'm going to play around with different techniques with a small subsample and see what gets me the best results. I didn't realize that it was so important to dilute the original PCR products before using them in the second reaction. Now, I have been getting pretty weak bands in my first PCR reaction. In the second PCR reaction I tend to get strong bands (with the occasional double band). Even with the weak bands from the first reaction, is it advisable to dilute the PCR product before performing the second reaction? Thanks.

Leonard, from my experience, any DNA fragment could be a "world" and more DNA template doesn´t mean more PCR product always. I suggest you to make several dilutions (three minimal: poor diluted, intermediate, full diluted). Good luck!!

I even use a Taq mastermix including the gl loading buffer, and use it as a template for nested PCR, and it works perfectly fine without cleaning. The only thing, as mentionned, is to dilute it as much as you can. In my hands: 1:100.

Thanks for the advice, everyone. I'm new to the world of genetics and it's taking me some time to accept the fact that more is not necessarily better. I'm preparing a bunch of small amplicons for Next Gen sequencing, so I really need to make sure that my samples are clean and only contain the targeted sequence. I don't have access to a plate reader or a spectrophotometer, so my quality control will just consist of running the final product on a gel. If my final product shows a strong single band of the correct length I am going to call it successful. Any thoughts on this?

Hey guys, anyone ever tried using and exonuclease to clean up the primers left of the first reaction? I got this suggestion, but I am not sure if it is really necessary...

Usually you can amplify the product of the first PCR reactions without needing PCR clean up.

What about performing multiplex in Nested PCR. Can we try using 15 cycles using first set of primers and 25 cycles using second set of primers in the same tube?

In my experience it is not necesary to perform a clean-up step. If you are sure you are not going to be able to see the first PCR product on an agarose then, as it has been sugested by a coleage you should try several dilutions of the first product on the nested step

I did nested PCR by taking 5uL of 1st reaction and without diluting it. It worked fine and even i got bands t the expected size. although in 1st PCR using outer sense primers no bands were seen 2nd PCR with inner sense primers showed specific bands.

You could try with some dilutions, taking in count the intensity of your first band.If you can't see it, you can try without dilution, but if it is intense you need dilution, because you could obtaind multi-bands.

Hi everyone,

I am working on Bacterial 16S ribosomal RNA (16S rRNA) gene amplicon sequencing for microbiome and using Nested PCR, i used AMPure beads after the first round then i run the second round and i did not get any bands after second round but when i skip using beads, i have got bands so, can someone help me?

thank you very much,

samia