Hi, I'm screening Blastocystis sp. in human stool gDNA by using PCR with 18S rDNA specific primers. The results contained both (strong and weak band) positive and nothing in gel electrophoresis. So, I used the nothing one as the template for the next PCR and the results showed both smear and the positive one. That means in the nothing one, it still has the specific product but too less amount to see in the gel electrophoresis. If I want to concentrate them for purification, can I use the previous PCR product as the template for the next PCR and are there any ways to remove the smear results? Thank you.
Hi Tawatchai,
You could try to gel-purify the correct band and use that for another PCR. If you don't have much though it might not work. You can try instead to optimise your first PCR from the original gDNA by modifying the annealing temperature, the annealing time, the amount of gDNA you use, and also the number of cycles.
If you try to use the second PCR, which contains a smear as well as your correct band, as a template for a third PCR you will likely get worse results as all that smear will only get worse.
Alternatively, you could try doing a nested PCR in the second PCR, but I don't have experience doing nested PCR with 18S rDNA, so I'm not sure if your product is long enough for this method.
I hope this helps!
-Anna
Hi Tawatchai,
You could try to gel-purify the correct band and use that for another PCR. If you don't have much though it might not work. You can try instead to optimise your first PCR from the original gDNA by modifying the annealing temperature, the annealing time, the amount of gDNA you use, and also the number of cycles.
If you try to use the second PCR, which contains a smear as well as your correct band, as a template for a third PCR you will likely get worse results as all that smear will only get worse.
Alternatively, you could try doing a nested PCR in the second PCR, but I don't have experience doing nested PCR with 18S rDNA, so I'm not sure if your product is long enough for this method.
I hope this helps!
-Anna
Dear Anna
Thank you so much for your helpful suggestion. I will try to do it and my advisor, he also mentioned about the nested PCR.
Thank you
Hi, one strategy you can follow and see, it may give satisfactory results, as we did it also and it worked with us. Run all of your samples and GOIs along with your reference gene in single well each in a single PCR plate. And for this plate use good amount of cDNA like 100 ng and primers for all of your genes in 10 pmole at least..(but this can vary as per your sample type and RNA, cDNA quality, sample type). Run by normal program of 40 cycles. Then from each well of this product plate prepare the dilutions of PCR products like 1:10, 1:25 and 1:50 of all of your samples with all of your genes including the reference one. And then run successive plates for all the dilutions for all the samples in duplicates (triplicates preferred) and see which dilution of HKG gives best results so calculate other samples for that dilution only and disregard other dilutions. But for second plate, run always at low cycle numbers less than 30, and all primers should be maximum 5 pmoles would work fine . This optimization is laborious no doubt but definitely you will reach to some positive conclusion..especially for the low target cases. While the Nested PCR is another fine approach but you need to design primers and wait to arrive at your doorstep. You can work with the theme I just mentioned...Thanks...
As Anna suggests the answer is a nested pcr with primers inside your original ones. A word of caution. Nested pcr is very efficient so I would start with a small amount of template...1ul of a 1:100 dilution is plenty. remember every 10 cycles is 1000 times more product so I would set up tube pcrs with 30 cycles and remove a tube every 5 cycles to see where good amplification occurs. When there is too much amplification the product becomes the major component of the pcr and the primers are no longer in huge excess so sometimes the product anneals with other product molecules and you will get large fragments forming if you over amplify the original product but generally nested ( or even hemi nested) pcr works very well
I agree with Anna. Purify your band and than use -2 ul of that product for Nested PCR. Primers for your nested PCR should be inside of your 1st PCR so that you get enhanced specific product only. Use minimum PCR cycles.
try to elute the pcer product from gel and then use 4 ul of same for next pcr
I agree with Anna. If you are getting smear in second PCR that means it going to be worse in next step. And you cannot sure about the re-PCR product quality though quantity. If you are going to species identification its better to use fresh PCR product for your further studies. You can try to optimize the PCR to get good result or you have to go for nested PCR. Good Luck!
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