Dear All,
I am going to use EvaGreen®/ Bio-Rad system to detect two genes using two pairs of primers.
The first pair was successfully detected previously using EvaGreen® master mix.
It was detected at thermal cycler annealing temperature 51 degrees with a 99 bp amplicon. However, I would like to design multiplex and detect two genes using the EvaGreen® master mix.
The second gene has a master mix designed from BioRad and the recommended temperature is 58 degrees with an amplicon of 195 bp amplicons.
How could I use the same well in ddPCR to detect both? Which Temperature should I use in my multiplex assay?
Thanks all
Best
Use gradient PCR to see if both primer sets have an overlap in usable range in annealing temp. Small products are generally easier to amplify at a range of temps because the ramping from the annealing temp to 72 will often hit the desired annealing temp along the way.
If you don't have a gradient capable thermocycler, then you'll just have to test several temps in several PCR runs.
Good luck!
It is quite tricky as you have so varying annealing temperatures. First, you should check how your second PCR works with 51 C. If it is not fine, try to check both PCR in a range of temperatures from 51 to 58. If there is a temperature point where both PCR give satisfactory results, use it as an annealing temperature for multiples PCR. If your PCR produces desired fragments, but shows smears of non-specific amplification, try touchdown protocol (the first cycle with 10-12 C higher, than decrease by 1 C per cycle down to working annealing temperature, than remaining 20-30 cycles with constant temperature).
If these do not work, your two PCR are probably not suitable for multiplexing.
Use gradient PCR to see if both primer sets have an overlap in usable range in annealing temp. Small products are generally easier to amplify at a range of temps because the ramping from the annealing temp to 72 will often hit the desired annealing temp along the way.
If you don't have a gradient capable thermocycler, then you'll just have to test several temps in several PCR runs.
Good luck!
Dear all,
I am sharing my successful experiment where i used graident pcr to determine the best temperature for both pairs of primers and then I performed a successful 2D Evagreen ddpcr
Thanks all
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics