Dear Rawan, could you give some more detail on what your samples are now and as a control and with what you otimised it? You mention you optimised conditions before, (probably with a control template, a plasmid or so?) and now it is not working anymore. In this case, i would not modify the conditions for PCR too much.

Do you now  not get any more good results with your control template (which u originally used)? In this case it might be that the control DNA could be degraded partly (or also the primers). If on the other hand, the control works, but your new samples not, something might be wrong with the sample dna prep.

Alternatively, in general, though it seems a not so scientific advice,  when PCR stops working, sometimes it is good to use everything new and not look back.  ^.^

Greetings

D,

Dear Rawan

the first problem with faint product u can increase No. cycle so it lead to increase copy number of gene and dissolve the faint band .

and the second problem u can test the primer on positive DNA or positive stander and when u get the good result u can complete your work , but I increase TM (annealing temp) to remove the primer dimer and get optimal result

finally good luck 4 u darling

regards

israa  

I sure that the problem not about DNA degredation , cause first I faced this problem with two SNP from three , the third one give the new bands as before , and the samples are very new. 

I optimized the condition with controls & patients DNA samples and they work well ! 

About the no. of cycles I worked with 35 , I think its enough , is not it ? 

I tried to increase the extension time of cycles to 50 and 60 sec instead of 40 , but I didnot get the requied result ! 

Also I increased the annealing temperature to get red of primer dimer , but actually I lose my faints band and the primer dimer still ! 

Thanks for your advices :) 

Hej Rawan, again, me,

i am not sure i understod perfectly, but at least you could exclude dna degradation, which is very good, and also if you usually and with other primer combinations get good amplification at 35 cycles it is fine. Extending the extension time should not bring so much.

But do you include a positive control in the pcr for the amplicons that did not work now ? (like of the old samples u used before, e,g.) and they still work?). If yes, and other primers work, then maybe your  absence of amplification is actually true - due to that the primers do not bind because the target regions are just not conserved in the now tested samples? Maybe they are different for these regions. is it not possible?

If you have established a working programme with cycles and annealing and extension that give reproducible product, don't change it.

In negative controls and without the right template it is often seen that primer "dimers" accumulate, but that does not mean that it will be favoured over or interfere with a true amplification to my experience.

Greetings

D.

If I understand you correctly, your PCR was working at some point but now doesn't any more. If so, try to use new primer dilutions made from your stocks.

Dear

u should  increase the No. cycle to 40 to solve the faint band

and also maybe u get the stander positive to test the primer on it firstly and then test in all your DNA

maybe I want to see the pic of your PCR product to see the faint band

regards

 Thanks for your answers,  

Actually I didnot run positive control. 

Today I made new primer dilution , increased the cycles to 40 , and the extension time to 50 sec , and I tried two higher annealing tempreture.

The results are attached. 

Regards 

Your PCR seems to be specific. How much genomic DNA are you using in each reaction? May be you need to increase the amount of template.

Dear Rawan, 

Your primers looks specific. Few things leads faints bands

i. phenolic compounds in the sample: You can know it by  A26o/280. It should be 1.8 to 2.0, if you have pure DNA.

ii. Concentration of template: (a) If it is too low, you will get faint bands. In that case try to increase initial template or perform nested PCR. (b) too much of template. In this case you need to increase primers and dNTPs in the reaction mixture.

Hope it will work.

Good luck

Thanks for your comments , I will take it in considerations.

I used to add 4ul DNA sample to 1ul primer.

I have to points to ask about,

If I run 5 samples only two of them give pcr product , I repeat the same samples in other run , other two samples give( differ from that in the first run) PCR products and the rest not, How I can explain that ?

what about using DMSO in PCR rxn?

Regards, 

4µl DNA of which concentration?

Dear rawan

what the cons. of DNA in this samples??

maybe this cause lead to faint band

keep in mind the cons. & volume of DNA sample in this run of PCR

Did you analyze if your primers (F and R) are complementary between themselves?

If not, you can use this software: oligoanalyzer https://www.idtdna.com/calc/analyzer

Where you put the sequence of each primer and then analyze if they form primers dimers with itself and with the another primer, and even all those secondary structures such as hairpins with their delta G, all this taking into account the conditions of your PCR (concentrations of cations: Na, Mg, and the temperature of annealing)

I have the same problem during my RT PCR, I got faint band of my target gene and high primer diamer. I changed the primer concentration from 0.2 to 0.1 but it doesn't work. I also trying to increase the number of cycle. is their any recommendation.

Dear Charith Raj Adkar-Purusho, pertaining to your answer, may I know how to increase dNTPs and the primer? I used One Step RT-PCR kit(Bioline) and my sample is RNA

Thank you