Primer-dimers

[@GeneticsLessons]

Watch my new video: How to design Forward and Reverse Primers kzbin.info/www/bejne/hZOunYZ_ZpuWhc0

[@ScientySundar]

Is there any videos in your channel, that would teach me about western blot, please provide links

[@GeneticsLessons]

No yet but will add soon.

[@ScientySundar]

@@GeneticsLessons I'm eagerly waiting!!

[@talithaamanda1129]

hi i got primer dimers too in my electrophoresis result. do high DNA concentration affects this? i used 50 ng/ul concentration, band was appeared but not too thick. and then i tried with 2000 ng/ul, band appeared very thick but i found primer dimers. can you please give me explanation? thanks in advance

[@GeneticsLessons]

Yes primer dimers formation can be a result of high concentration of DNA and primers. Troubleshooting and Prevention: - Review your primer design to ensure they do not have extensive complementary regions. - Optimize the PCR reaction conditions, especially the annealing temperature, to promote specific binding of primers to the target template. - Use primer concentrations within recommended ranges. -If primer dimers persist, you can consider adding a "hot start" step to your PCR protocol. This involves temporarily deactivating one of the PCR components (usually the enzyme) before cycling begins, preventing nonspecific amplification during the initial setup. Remember that some primer-dimer formation can be expected, but their presence should be minimized to avoid interfering with the accurate interpretation of your results. Optimization and careful experimental design are key to obtaining clear and reliable PCR and electrophoresis outcomes.

[@talithaamanda1129]

@@GeneticsLessons thank you so much! do you have recommendation for DNA template and primer concentration?😊

[@GeneticsLessons]

- Start with an appropriate amount of DNA template. Generally, template concentrations of 10-100 ng/μl are recommended for most PCR reactions. - Follow the manufacturer's recommendations for primer concentrations provided with the PCR kit or primer synthesis service you are using. - Ensure that the concentrations of both forward and reverse primers are balanced. Use equal concentrations of both primers, typically ranging from 100 nM to 500 nM each.

[@GeneticsLessons]

Primer-dimer formation can be influenced by the annealing temperature. A higher annealing temperature can reduce primer-dimer formation, but it should be within the range that allows specific primer binding to your template.

[@talithaamanda1129]

@@GeneticsLessons thank you, really appreciate your explanation, i'll try again tomorrow. have a great day😊