Hi there,
Are you wondering why you can't see any band of genomic DNA on gel or why don't you get any band amplified during PCR?
Dear Atefe.
Absence of a band may be attributed to the super coiled structure, in which case the primers/polymerase will not be able to access the required sites.
Regards
I agree with Maria. Try increasing your PCR cycles to 40. If you still don't get a band, try using primers that you know will amplify the DNA you've extracted (i.e. old valid primers in your freezer or primers that amplify housekeeping genes) to check if the DNA is amplify-able. If you then still don't get a band, replace the reagents of the PCR reaction one by one (including the water), until you get a subsequent band. And if that doesn't work, you should should consider changing the DNA extraction protocol. If all these methods still fail to give results, then you should use a different PCR machine. This is the order in which I would troubleshoot PCR failures, and it does not include covering everything that can go wrong, but most of the time, these steps will allow you to isolate the problem.
Atefe,
Maria and Werner provide good answers, above. I would follow Werner's suggestions, step by step.
However, your question sounds like you are expecting to see a band of your genomic (target) DNA. Generally, the target DNA concentration can be so low that you would not easily see it on a gel (depending on how much you load onto the gel). You don't need much target DNA in order for PCR to work.
If your PCR did not work, normally you would not expect to see any bands at all (expect possibly faint bands of unused primers). What is the concentration of your template DNA? If you use, say, about 10 ng of total template DNA, and you run PCR reaction, and then load, perhaps 20% of your PCR reaction onto a gel (maybe 5 to 10 uL), then you are only adding 2 ng of template DNA onto your gel. That might not be enough to visualize, depending on thickness and density of your gel, visualization method, etc.
Thank you for all of your comments.
This problem was solved by adding Dmso.
Is evidence of a specific defect in the target gene in the event that the study to show a certain effect
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