Hey All,
How i can avoid or prevent Primers dimer during PCR amplification? I Know about the solutions that creating optimals primers and using proper primers concentration, also to change or adjust the annealing temperature.
But Iam looking for another solutions can use it to Helps us .
Second, what the best application can helps me to to check PCR primers ( Free app).
Thanks,
Use any hot start polymerase and it will help minimise primer dimer. Using less primer is also very helpful.
Primer 3 and primerblast are very useful in checkinh primers
Primer3Plus at www.primer3plus.com
The primer analysis tool at IDT is also very useful
https://eu.idtdna.com/pages/tools/oligoanalyzer?returnurl=%2Fcalc%2Fanalyzer
Thermo fisher also has a nice tool (https://www.thermofisher.com/uk/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html).
DMSO or mgcl2 can be used for getting better results. Conformation through primer blast can also be useful
Yasmin Yosif In my work, I encountered the same problem and the first reason was attributed to the high degree of annealing temperature. The most suitable method I used to determine the optimal temperature at which the bands become evident without Primer’s dimer was as follows: I performed a bulk of samples, then I conducted gradient PCR, I relied on this website
https://www.thermofisher.com/eg/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html
The second reason was used too much primer was added/ primer concentration was too high so, you should titrate the amount of primers for your system.
You have to test them online before ordering ! by using PrimerQuest or Primer3 ..these software will give the results (good or not )
Basically in the following ways:
1. design primers to reduce dimer probability, such as using primer-blast: https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?GROUP_TARGET=on
2. reduce primer concentration
3. use hot start
https://lab.plygenind.com/how-to-avoid-primer-dimers-in-pcr-reaction
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