Another unpopular cause of weak bands would be due to the use of impure nuclease free water (e.g. you use water that you've kept in a tube for some time which might be contaminated) and I have experienced this myself. If all of your reagents and conditions worked fine beforehand, you might want to try using a fresh tube of nuclease free water in your subsequent experiments. Contaminated or incorrectly designed primers may also affect your PCR bands.
Other troubleshooting suggestions may advise you to fine-tune your annealing and denaturation temperatures or adjust your DNA concentrations. I have attached some links that I found useful when I encountered problems in conducting my own PCR reactions.
- http://www.bio-rad.com/en-sg/applications-technologies/pcr-troubleshooting
- https://www.fws.gov/aah/PDF/PCRTS1_NoBand.pdf
Best of luck!
Dear Shima,
I think you should examine PCR troubleshooting book that could find anywhere. If you get only weak band, you should increase your DNA amount or cycles number. If you have smear, you should calculate your DNA purity and you should get more pure DNA extraction. And more of them, when you examine troubleshooting book, get more answer for your question.
Another unpopular cause of weak bands would be due to the use of impure nuclease free water (e.g. you use water that you've kept in a tube for some time which might be contaminated) and I have experienced this myself. If all of your reagents and conditions worked fine beforehand, you might want to try using a fresh tube of nuclease free water in your subsequent experiments. Contaminated or incorrectly designed primers may also affect your PCR bands.
Other troubleshooting suggestions may advise you to fine-tune your annealing and denaturation temperatures or adjust your DNA concentrations. I have attached some links that I found useful when I encountered problems in conducting my own PCR reactions.
- http://www.bio-rad.com/en-sg/applications-technologies/pcr-troubleshooting
- https://www.fws.gov/aah/PDF/PCRTS1_NoBand.pdf
Best of luck!
Old PCR primers aliquote that you were using might be one of the reasons of getting faint bands. Because, repeated freeze and thaw of primers degrades the quality of primers. And the second most common reason is the template DNA. If possible get a fresh template DNA and go with the same PCR conditions. Good luck.
If the primers were dissolved in water not TE then acid depurination of the oligos from CO2 absorption from the air may have degraded the primers. Order new primers and dissolve in TE
Hi @Andrew Octavian Sasmita,
Thank you so much for sharing the links. Do you happen to have the part II of the link below? I tried to find it online but to no avail. Is it available yet? Thank you very much, cheers.
- https://www.fws.gov/aah/PDF/PCRTS1_NoBand.pdf
Dear @Yong Kian Lim,
Sadly, I don't think the subsequent parts are available as I also tried to find a complete set of chapters and not just the one. You can tune into their website, although the content might differ: https://midsci.com/news/pcr-troubleshooting-guide
Hi, Shima,
I agree to everyone's recommendations above especially on the use of pure reagents and the integrity of your primers. Answering in the context of the information you provided, faint bands of PCR product might come from various sources such as possible PCR contamination. You may refer to the following links for more information:
https://tools.thermofisher.com/content/sfs/manuals/MAN0012617_Guidelines_Preventing_Contamination_PCR_UG.pdf
https://www.promega.com/-/media/files/resources/profiles-in-dna/802/identifying-and-preventing-dna-contamination-in-a-dna-typing-laboratory.pdf?la=en
- Badges
- Science method