Hello.
I tried to get bands from a few samples using the nested-PCR method, and I tested the 2nd round PCR with 0, 10, 100 and 1000 dilutions of the first product. Only in 0 dilution, smear is seen. Where is the problem with this test and how can I optimize this test?
Are you getting bands of amplified product from the first round pcr before reamplification?. If you are getting a primer dimer this could be spoiling the second round pcr so it might be worth exo-sapping the first round product before reamplification. If there are bands at pcr1 then the smear will be over amplification of the pcr product and amplimer concatenation. If there is no band in pcr1 then I would lower the annealing temperature 2-4c and then try pcr2 with 1:100 and 1:1000 dilutions (1ul and 25 cycles). If the pcr is fully nested you could try a 1:100 dilution of your first round product and then 2 round hemi nested pcr of outerf-inner R and innerF - outerR in case one of the inner primers is not working due to the presence of a snp under the primer
Paul Rutland
Thanks. I sent the gel electrophoresis of dilution in 2nd PCR round.
There is no primer-dimer and any band in 1nd PCR.
But when I diluted the first product, no band was seen (1:100 and 1:1000) (in pic).
Could DNA have been destroyed?
Thank you Fatemeh Hosseini . I assume that 10b is a positive control in which case the annealing temperatures are correct and the pcr is working. It still looks to me as if the undiluted samples may still be overamplifying so it would be interesting to see the amplification at some dilutions between 0 and 1:100 with the 2 internal primers as well as the hemi nesting with one first round and one second round primer ( both orientations)
Is 10b a clinical positive sample or is it amplified from purified dna sample ( I am looking for any difference between 10b and the other samples that may be interfering with the first round pcr in the19-23 samples
Yes, 10b is a DNA extracted from the blood. and I so think that anealing tempperatures is ok. But why other samples are negative with 3 dilution and have smear without it?
Could there be a need for more dilution?
It may be that all of the other samples are negative and so there should be no amplification and that the smear at no dilution is non specific amplification
It might also be that the 19-23 samples are made in a different purification process and that some pcr inhibitors are left in the purified dna from these samples. You could test this idea by running 10b again and also 2 of the samples both with, and without, adding the same amount of 10b dna to the tube with 19 or 20 dna so you run 19 and 20 and also 19+10b and 20+10b. If your dna samples have pcr inhibitors then these woeking PCRs will fail and that is a useful diagnostic test
Hi, Fatemeh and Paul.
Long time I've been out of researchgate. Very pleased to see Dr Paul around still helping us all.
There is a paper from Han (Article Cause and solutions to the polymerase chain reaction smear p...
) who has shown that If you keep working with nested PCRs for sometime, your primers and reagents get so contaminated that starts to build up smears as results. So, my question is: Are you still using the same primers for a long time and keep these reagents in the same freezer? If so, Maybe the same is happening to your reagents. His solution was to move the primers a little out of the previous area. Just a pinpoint.Another option, depending of what are you looking for, may be to perform a booster PCR. So, similarly to a Nested one, you will dilute the first PCR amplicon and perform the same PCR once again with the diluted amplicon and see If it gives you a band or a few bands with the one you want on it.
Hope it helps.
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