Theoretically, is there a problem with running a PCR and then running the same system again, with the same primers, with the product of the first reaction to further amplify the product? The primer binding site should still be there. Maybe it would be wise to lower the annealing temperature a bit?
As Artur says, it is definitely possible to re-amplify. You can have an idea of how the re-amplification might works from how the first amplification looks like on the gel.
If the lane is clean, completely empty, you have chances of obtaining something specific in the second amplification (as Artur says, if you get anything at all). In this case even using 1-2 ul from the first PCR can work (we use this trick to genotype single mouse blastocysts, if you have an idea what they are and how little DNA we can extract from them).
If the lane is already showing several weak bands (most likely your DNA will make a smear as background, anyway), almost certainly you have no chances of amplyfying specifically. But in that case I would suggest to increase the annealing temperature in the first PCR and lower it in the second (a sort of step-down PCR, but in two PCR rounds).
Still, nested PCR is safer!
Good luck!
It depends on which material you work. For plant tissues a direct-PCR can give nice products at once, ommiting laborious extraction methodology, starting from minimal amount of material, and presenting good specificity thanks to the hot start polymerase used in the kit.
Yes, it should work. If you have a good PCR product in your first reaction you can use the product as a target for another reaction but if you have problem with your 1st PCR it is not recommended. Try to purify your template.
Although theoretically possible you should do all your best to avoid it, because, as Artur and Pietro pointed out, you often end-up amplifying short aspecific products (e.g. primer dimers). A trick you can use when moving nested primers away is technically difficult, is to just shift their position a few bases away from the first set. Often just 3-4 bases are enough to greatly improve the specificity (and therefore the efficiency) of your reaction. Of course you must shift the 5' nested primer towards the 3' and vice versa.
For Viral nested PCR's it is advised to use both nested primers.
I worked on HEV (RNA virus) and Human Parvovirus B19 (DNA virus), both gives good amplification in second round product.
I will retest the outer primers and keep posted the results.
If the first round PCR product is single amplicon, although very faint band, and if all the cycling conditions are good, it perfectly amplify viral Nested PCR also. I just got the perfect product in second round in viral RT PCR.
Best wishes.
- Badges
- Science method