PCR is inconsistent - (Feb/04/2018 )

Hi everyone, I need your help regarding my pcr problem. So early last week I created a serial dilution of a purified linearized plasmid (around 4 kb). I performed a gradient pcr on one of the dilution solution to determine the optimize annealing temperature. All was done and well.

Next, I did the pcr with the optimized Ta on all of the serial dilution. There was also no problem. I got much PCR product on every samples. On the next day, I wanted to reproduce the result. But this time, there was only smearing in the agarose with the exception of the highest concentrated solution. I tried the pcr again but the weird thing was that the highest concenctrated solution did not show any more band but the second highest one did. The third try: band on the highest and the second highest but still no product on the rest of the samples. How could it happen?? I aliquoted the diluted samples and stored it at -80C, so it shouldn't be degraded that fast!

I'm now running a positive control to test the dNTP, buffer, and polymerase. But if the positive control work but the samples don't, what do you suggest is happening? Is it possible that the primers are degraded? We store the stock solution at -80C and the working solution was freshly made (stored at -20C).

I need this PCR to work before thursday... so any help/idea is appreciated. Thank you

I wouldn't free thaw my DNA samples from that high temperature. Ideally, if you want to use your samples the next day, you should store them at 4°C (for 2-3 days). I have used this method with success. If I want to store it for a month or so, I keep it at -20°C. And if I want to keep it for months or years, I store them at -20°C. 

If the +ve control works, then your samples have degraded. Again, I store my working primer stock at 4°C for a week. I make enough to last for a week and I store a batch of working concentration at -20°C. This way, I avoid repeated freeze thaw of the stock. 

Did you try measuring the DNA samples on a nanodrop? Do the values vary from the original? How is the A260/230 and A280/230 values?

Hope this helps.

Hi,

The material that I am using is cDNA from rice. generally when we extract RNA and convert it to cDNA we get an amplification at 26 cycles, but in my case I did not get any amplification at 26X except in 2 samples, but got amplification at 35x. Now when i go in for normalization and change the volume (concentration) of cDNA that i take for keeping a PCR, not all samples get amplified. For eg. suppose I have taken 1 ul of cDNA initially to keep a PCR with actin all gets amplified at 35x, but when I change the volume of cDNA from 1 to 1.5 or 0.8 ul they don't get amplified even at 35 x. all other components are taken according to the instructions of the company. I know my template is very low, but is there a solution for this kind of inconsistency?

If you are seeing amplification at 35 cycles then you are amplifying very very tiny amounts of DNA - literally 1-2 copies per reaction. Now imagine you had a tube/well where by chance, because of the low abundance, you didn't add any copies, even though you added the sample... This is the reason you get inconsistency - some of your wells will by chance not contain any target DNA. The only way to overcome this is to add more sample.

Thank you. Let me try increasing the sample.