We are working on Biofilm and Quorum sensing genes of E.coli, we suggest some of reasons maybe:- Contamination, Annealing Temperature, Melting Temperature, DNA Extraction mistakes in the Techniques, we identified them by biochemical tests to be sure the isolates are E.coli, but now we are collecting samples from the beginning and doing the procedure again
Too much or too little DNA can lead to no bands. Use approximately 0.5 ng – 0.5 µg of total genomic DNA per 25 µl reaction.
If the DNA quality is poor, it may not amplify bands
Try diluting DNA
Or
If you get no bands for any DNAs, you likely have a problem with your primers.
Make a new working dilution from your concentrated stock
Primers can degrade when maintained at lower working concentrations or following repeat freeze/thaws.
Re-order primers
The annealing temperature is a critical parameter in PCR, as it must be set correctly for the primers to bind efficiently to the template DNA. If the annealing temperature is too high or too low, the primers may not bind effectively, and the PCR may not produce a band.
A lot of situations can be happening there, it could be that you are not getting the DNA ratio correct, if that you can concentrate your sample or dilute it, another thing is that you could check is contamination, if you have contaminated water, primers or polymerase, or if your extraction is not performed correctly, that could lead to other stuff getting into the "purified" DNA sample, is really a tricky situation, what i would do is try doing another method for extracting DNA, verify contamination or checking the temperatures used in the PCR.
I would start with a PCR reaction with a very low annealing temperature. If no bands appear, you can still do a lot of troubleshooting. Do you see the band of the primers at the bottom of the gel? Try running different amounts: sometimes you buy a pair of primers but one of them has much less than the other. You also need some positive control: using the same reaction conditions, just trying to amplify some other gene with different primers. That way you can know if your DNA is OK. Or, you can use different bacterial DNA to see if there is amplification. When purifying DNA make sure you don't leave ethanol in the pellet, because it can inhibit the PCR reaction. If the positive controls don't show a signal, you can then make new solutions: use new buffer, MgCl2, dNTPs, etc. Good luck!
There could be several possible reasons why no bands are observed in the PCR amplification of 16S rRNA of E.coli genes:
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
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