I am doing recombinase polymerase amplification primer design now.
Can I ask anyone what kind of product size is the best? about 200 bp?
What is the need to consider?
Primer GC%? Primer Size? Primer Tm?
THX
Hi,
Are you going to do isthermal amplification using RPA kit? if so, you can choose the product size 170-200 bp. Annealing should be 37 to 40 degree centigrade.
best of luck.
The general rule of thumb with RPA is that shorter amplicons amplify faster. There is no thermal melting or annealing, so don't worry about Primer Tm, that's what the recombinase proteins are there for. You can use PCR-length primers, but for best results 30-35mer primers are best as they bind more recombinases and form longer recombination filaments. There are guidelines for assay design on the TwistDx webpage that might be useful:
https://www.twistdx.co.uk/en/support/rpa-assay-design-2
Otherwise, have you emailed [email protected] to see if they can answer your question?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology