My PCR is alwayes negative result on the gel. Although the concentration of DNA is high in my samples when I measured it , I putted 4ul of sample in the reaction mix with negative result .I will try to dilute the sample 1:10 and use the same volume,, is this right step?
The most likely cause for your negative result is too high DNA concentration. Best would be to make serial dilutions (2-fold steps), at least use several dilutions such as 1:10, 1:30 and 1:100.
Hi,
Below is a link for PCR Troubleshooting Guide. I hope it will help
https://midsci.com/news/pcr-troubleshooting-guide
Good luck
Soha,
What is the concentration of your DNA? and also the quality is good? As Muhammad said, it is not only the DNA, but also other factors. Sometimes the primers from literature are not good at all, it is worth to check if that article has been cited a couple of times for the PCR protocol so that means that the protocol referred in the article not only works in the hands of the authors.
Soha,
Like the people above said, it might be the concentration or the purity of your DNA. But it might also be some other inhibitor. Try adding BSA, because it helps with the annealing, making it more specific. Also the Tm might not be okai. Always use a positive and a negativ control when you set up a PCR. Furthermore, even the polymerase can inhibit the reaction.
Try changing one thing at a time, not everything at once, because this way you can figure out exactly what works.
Best of luck!