i want to confirm my finding regarding Ancylostoma Caninum eggs by molecular studies, which is ``ITS-2 amplification``.
but the number of eggs in my samples is not to much and i cant get any band from my PCR.
what is your suggestion?
If you are amplifying single cell dna then either use whole genome amplification of the original dna to increase the amount of template or use nested pcr which is very good for producing a clean product after many pcr cycles. Just running more pcr cycles with a single primer pair usually leads to an increase in non specific amplimers. is your positive control sample amplifying?
Dear Roxana Nezami
Can you send the DNA gel picture? Coz., for PCR even a minute quantity of DNA is enough for amplification. Either you need to optimize your PCR conditions (like optimizing annealing temperature) or go for undiluted gDNA.
Cheers
You must be quantifying your samples for the DNA before PCR or are you directly hopping over to PCR?
Paul Rutland at first i try to extract the DNA from the fecal samples. and in some cases the number of eggs is very low. maybe my problem is in this stage that i can't get enough gDNA.
i tried nested pcr but i didnt get any results.
Abhijeet Singh always by using nanodrop, I quantify the amount of the DNA, but I'm not sure this amount is only for my eggs.
Hi Roxana,
nested pcr is incredibly sensitive so I find no amplification worrying and wonder if there may be a problem elsewhere in the pcr protocol. Were both of your nested primers inside the first round primer set..Did you test the nested primer sets on a guaranteed positive control sample that will amplify and dilutions of that sample?
Often it is the case that nested amplifications are heavily overamplified and instead of a strong single band you get a long smear of concatenated product but no single band.. A regime something like 30 cycles of 1st round pcr then dilute the pcr mix 1;100 and amplify 1ul of the 1/100 for somewhere between 15 and 25 cycles to avoid over amplification.
It is also possible that the dna from fecal samples contains pcr inhibitors so strangely amplifying less dna ( therefore less inhibitor) amplifies much better than more dna. Have you had any sample amplifying well enough that you can use as a positive control sample?
Paul Rutland Thank you very much for your complete explanation.
Before I tried with one of the positive samples and I got a very weak band.
and today after changing the amount of materials and adding Mgcl2 I saw some bands but not in the right place.
what is your opinion about this finding?
Thank you in advance
Sushmitha T J i tried Gradient PCR to find the best annealing temperature, and here it is my today finding.
Roxana Nezami
I cannot see an amplified product on this gel. The diffuse bands at less than 100bp on the gel are primer dimer (PD). PD forms when a few bases of one primer anneal to complementary bases on the other primer and form a short double stranded product. This melts easily and has complete homology with the primers so amplifies very well and will eventually remove all of the primer in the reaction resulting in no amplification..
My feeling is that only a tiny amount of your dna is originating from the eggs so you need to investigate nested pcr further. If this picture is the amplification of the outer primers then use less primer and ideally a hot start polymerase to minimise PD and run 30 cycles of pcr which will probably not show any amplification because the amount of egg dna is so small. Then take 1ul of this reaction and dilute it 1in 10 in water. Then re amplify 1ul of diluted invisible product and run 25 cycles of pcr with the internal primers. Nahed Hussien
answer is a good one. Touchdown pcr will minimise primer dimer and usually gives a good product (clean) which can be re amplified.While testing samples you may want to look up dna preparation from your parasite eggs to make sure that your dna preparation method is the best for getting dna from the eggs just in case they are hard to lyse so making release of dna inefficient
It may be that the amount of other dna is inhibiting pcr and if this is the case it may be worth trying to separate the eggs from the bulk of the fecal matter before doing the dna prep but parasitology is not my area so read/ask around to see if people find it necessary
Paul Rutland Yes exactly, the amount of eggs is very low.
So, I use this PCR product for nested PCR and amplification with specific primers for Ac ITS-2.
I hope I can have some results.
Thank you again for your explanation and time
I hope, you get visible bands in your genomic DNA isolation. If yes, you can try all the possibilities discussed above.
You can also try adding BSA and DMSO in your PCR master mix to enhance your reaction.
All the best.
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