Hello!
Not completely specific primers and suboptimal reaction conditions (annealing temperature, magnesium ion concentration, Taq polymerase concentration, primers concentration are the main reasons for the appearance of non-specific PCR products
https://www.jax.org/jax-mice-and-services/customer-support/technical-support/genotyping/troubleshooting/artifact-band#:~:text=Artifact%20or%20non%2Dspecific%20bands,such%20bands%20can%20be%20disconcerting.
https://www.bio-rad.com/en-ma/applications-technologies/pcr-troubleshooting?ID=LUSO3HC4S#gel2
1.Anealing temperature can be one reason.
2.Make conditions of your PCR programm more stringent.
3. Make sure your primers are well designed, if your gene of interest have multiple sequence repetition and primers are on those repetitive regions it can give you non specific product.
4. Make sure there is no contamination in your sample, for that do a PCR with positive and negative control and run on agarose gel along with your (pcr) samples with ladder.
5. If you are using master mix confirm if it is contaminated or not.
6. If making cocktail for PCR increase amount of Mgcl2, it can help.
7. Its a very common problem in PCR if you are optimizing the anealing temperature of your primers make conditions stringent.
(For example your primers Tm is 58°C try somewhere around 60°C, 61°C.)
Hope this helps
Good luck.
Make sure if they are non specific band or you are confusing the bands with dimers if the product size is small.
You will get a specific answer if you share the gel picture.
Proper designing of the primer, as per the region of interest of your study.
Optimization of annealing temperature during PCR for the designed primer.
Optimization of the amount of MgCl2, which otherwise can trigger random and rapid annealing to unspecific nucleotides.
Be careful while handling naked DNA for contamination.
Check if the other reagents are contaminated or too old. Avoid using them, as they lose their activity.
Hope it helps.
All the best.
To add to Sana Shamshad 's comment, I would also suggest you do a gradient PCR to find the correct annealing temperature for your primer set.
For example for calculated 58°C run a gradient assay from 52°C to 62°C
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