Hello All
I have a question regarding real-time (RT) Primers Optimization. We usually perform gradient-PCR with standard PCR conditions for primer optimization for qualitative PCR. But for RT-primers optimization, what changes should I make? I put on standard PCR conditions but I got non-specific bands.
Can anyone help me in this regard?
Optimizing primers for real-time PCR (RT-PCR) can be done through various methods, and gradient PCR is one of them. However, it is not always necessary to use gradient PCR for RT-primer optimization.
One approach for RT-primer optimization is to use a standard PCR protocol and a range of annealing temperatures, just like in gradient PCR. However, since RT-PCR is more sensitive than conventional PCR, it is important to use lower annealing temperatures and shorter annealing times to avoid non-specific amplification. A good starting point for annealing temperature can be calculated using the Tm of the primers, but optimization may still be necessary.
Other methods for RT-primer optimization include designing primers with specific melting temperatures (Tm) or using software programs to predict the optimal annealing temperature. Some software programs, such as Primer3 and OligoCalc, also take into account the specific conditions of the RT-PCR reaction, such as the presence of reverse transcriptase and the concentration of cDNA template.
While gradient PCR can be used for RT-primer optimization, it is not the only approach. It is important to consider the sensitivity of RT-PCR and adjust the annealing temperature and time accordingly to avoid non-specific amplification. Additionally, software programs can aid in primer design and optimization.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
Article The Ultimate qPCR Experiment: Producing Publication Quality,...
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating