We have been characterizing our transgenic mice lines as a routine job in lab for years and It has been working fine until a recent batch of primers for VEGF has arrived. We started observing streaks in gel instead of proper band, or at times no reaction at all.
changed the primers for 2-3 times already, water, cycles of PCR.
I really don’t know what else could go wrong.
High template concentration may also cause such smearing, so try with reduced concentration of template DNA. Sometimes higher concentration of Primers cause such issue.
After you rule of the concentration issue of both of these, please rerun a annealing temperature gradient.
please change everything and start fresh. it means you have to reorder new oligos also.
It happens with everyone, sometimes usual protocol may not work.
Hi! Have your check your DNA? Is your DNA working with different primers?? To discard that is DNA problem, you can check primers of a housekeeping gene (or other primers that are working) and see if they work with your DNA.
It has happened to me that i have some primers working well for some time and then I only see smear. Then I have tested my DNA with other primers and is fine. Then I have re ordered primers and they still produce smear. There is not a clear explanation for this...but it seems that some primers are just unstable and unreliable. A solution might be to design new primers.
Can we see an image of a typical streaky gel please?
Is there a streak or any sign of amplification in your no template control?
You may have post pcr contamination.
https://blog.benchsci.com/5-steps-for-mouse-genotyping-and-troubleshooting-tips
Ashok Kumar I did change primers and mastermix I use in lab is from Apex, which had been working fine. And as I mentioned the DNA is working fine for the other primers pretty well.
Diana Duarte-Delgado yes it is working with other primers pretty well and I did change the DNA as well a couple of times.
Ok, then I think you can try to re-design the primers... is difficult to explain why the primers stopped working but I think now the only solution is to work with other primers. As I told you, this also has happened to me and I work with bread wheat.
High template concentration may also cause such smearing, so try with reduced concentration of template DNA. Sometimes higher concentration of Primers cause such issue.
After you rule of the concentration issue of both of these, please rerun a annealing temperature gradient.
redesigning primers is last option.As I have faced the same situation in my experiments.
Hi Gaurav,
I always recomment this paper for general PCR optimization:
Article Multiplex PCR: Critical Parameters and Step-by-Step Protocol
But i your case I would redesign the primers as Diana Duarte-Delgado proposed.
Thank you all for your feedback on the my issue, I am going to try all of these and update when it works.
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